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WO2024249846A2 - Analyse épigénomique d'échantillons fixés au formol et inclus en paraffine - Google Patents

Analyse épigénomique d'échantillons fixés au formol et inclus en paraffine Download PDF

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WO2024249846A2
WO2024249846A2 PCT/US2024/031983 US2024031983W WO2024249846A2 WO 2024249846 A2 WO2024249846 A2 WO 2024249846A2 US 2024031983 W US2024031983 W US 2024031983W WO 2024249846 A2 WO2024249846 A2 WO 2024249846A2
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chromatin
sample
protein
rnapii
dna
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Steve HENIKOFF
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Fred Hutchinson Cancer Center
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • the invention relates to assays for detecting and/or quantitating sites of DNA accessibility in chromatin in formalin-fixed paraffin-embedded (FFPE) samples.
  • the invention further relates to methods of using the assay for epigenomic profiling of FFPE samples.
  • FFPE formalin-fixed paraffin-embedded
  • chromatin profiling has the potential of identifying causal regulatory element changes that drive disease.
  • the prospect of applying chromatin profiling to distinguish regulatory element changes is especially attractive for translational cancer research, insofar as misregulation of promoters and enhancers in cancer can provide diagnostic information and may be targeted for therapy (Armstrong, S. A., Henikoff, S. & Vakoc, C. R. Chromatin Deregulation in Cancer (Cold Spring Harbor Press, 2017)).
  • chromatin profiling techniques to FFPEs (Amatori, S. & Fanelli, M.
  • Chromatin Immunoprecipitation from FFPE tissues. IntJ Mol. Sci. 23, 1103 (2022)). Although several methods have been developed for chromatin immunoprecipitation with sequencing (ChlP-seq) using FFPEs (See, e.g., Kaneko, S. et al. Genome-wide chromatin analysis of FFPE tissues using a dualarm robot with clinical potential. Cancers (Basel) 13, 2126 (2021); Font-Tello, A. et al. FiTAc-seq: fixed-tissue ChlP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues. Nat. Protoc. 15, 2503-2518 (2020); Amatori, S. et al. Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology. Clin.
  • ChlP-seq for chromatin profiling include ATAC-seq (Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213-1218 (2013)), DNase-seq (Jin, W.
  • FFPE-ATAC A highly sensitive method for profiling chromatin accessibility in formalin-fixed paraffin-embedded samples. Curr. Protoc. 2, e535 (2022); Zhang, H. et al. Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples. Genome Res. 32, 150-161 (2022)).
  • the same group also similarly modified CUT&Tag and included an epitope retrieval step using ionic detergents and elevated temperatures, which they termed FFPE tissue with Antibody- guided Chromatin Tagmentation with sequencing (FACT-seq) (Zhao, L. et al. FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.
  • RNA sequencing enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms
  • FFPE tissues which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis using RNA. Exposure of tissue to ⁇ 4% formaldehyde for days badly damages RNA and DNA and causes cross-links to form between tightly bound proteins and nucleic acids. The RNA obtained from such samples is often badly degraded, fragmented, and chemically modified, which leads to suboptimal sequencing while DNA is better preserved.
  • Embodiments of the present invention are based, in part, on the development of assays for chromatin profiling of FFPE samples, allowing for simultaneous chromatin profiling and accessibility mapping in FFPE samples with improved signal to noise at a low cost and improved speed compared to the current state of the art assays.
  • Embodiments of the present invention are also based, in part, on assays using RNA polymerase II (RNAPII) profiling in FFPE samples to map the transcriptional machinery itself directly on the DNA regulatory elements to obtain direct measurements of transcription activity, including nascent transcription.
  • RNAPII RNA polymerase II
  • an in situ method of mapping the location of a protein on chromatin in a cell from a FFPE sample comprising treating the FFPE sample to remove the paraffin; permeabilizing the sample; contacting the sample with a first affinity reagent that specifically binds to a targeted chromatin protein, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules; excising the tagged DNA segment associated with the targeted chromatin protein; and determining the nucleotide sequence of the excised tagged DNA segment, thereby mapping the genomic location of the targeted protein on chromatin.
  • a DNA-based in situ method for measuring transcription in a cell from a FFPE sample comprising: treating the FFPE sample to remove the paraffin; permeabilizing the sample; contacting the sample with a first affinity reagent that specifically binds to a protein involved in transcription regulation, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules; excising the tagged DNA segment associated with the protein involved in transcription regulation; and determining the nucleotide sequence of the excised tagged DNA segment, thereby mapping transcriptional activity on chromatin.
  • methods of monitoring a disease or disorder comprising performing a method as described herein on samples obtained at two or more points in time from the same subject, and comparing an amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin in each sample to a reference and/or to each other.
  • the disclosure provides a method of diagnosing a disease or disorder in a subject, comprising performing a method as described herein on a sample from the subject, and diagnosing the subject as having the disease or disorder based on an amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin to thereby diagnose the subject as having the disease or disorder.
  • a method of prognosing a disease or disorder in a subject comprising performing a method as described herein on a sample from the subject, and prognosing the disease or disorder in the subject based on the amount and/or genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin.
  • the disclosure provides a method of detecting hypertranscription in a sample, comprising performing a method as described herein, wherein an increased amount of transcriptional activity on chromatin thereby detects hypertranscription in the sample.
  • the disclosure provides a method of quantifying increases or decreases in RNAPII over a plurality of loci, comprising performing a method as described herein, wherein the first affinity reagent is an affinity reagent specific for RNAPII, e.g., a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2/5, or RNAPII-Ser5/7, and further comprising comparing the results to a control reference.
  • the first affinity reagent is an affinity reagent specific for RNAPII, e.g., a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2/5, or RNAPII-Ser5/7, and further comprising comparing the results to a control reference.
  • the disclosure provides a method of detecting presence of a protein of interest on chromatin, comprising performing a method as described herein, wherein the first affinity reagent that specifically binds to the targeted chromatin protein is specific for the protein of interest to thereby detect the presence of the protein of interest on chromatin.
  • the disclosure provides a method of detecting an amount of a protein of interest on chromatin, comprising performing a method as described herein, wherein the first affinity reagent that specifically binds to the targeted chromatin protein is specific for the protein of interest to thereby detect the amount of the protein of interest on chromatin.
  • the disclosure provides a method of detecting an epigenetic modification on a protein, comprising performing a method as described herein, to determine the presence of the epigenetic modification on the protein.
  • the disclosure provides a composition comprising a deparaffinized and permeabilized FFPE sample containing an RNAPII-specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions.
  • the disclosure provides a composition comprising a deparaffinized and permeabilized FFPE sample containing a chromatin protein specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions.
  • the disclosure provides a kit comprising two or more reagents selected from a RNAPII-specific affinity reagent, one or more chromatin protein-specific affinity reagent, a SDS solution, a Triton® X-100 (octyl phenol ethoxylate) solution, a transposase solution, a tagmentation buffer, a cross-linking reversal solution, and amine- functionalized magnetic beads.
  • a kit comprising two or more reagents selected from a RNAPII-specific affinity reagent, one or more chromatin protein-specific affinity reagent, a SDS solution, a Triton® X-100 (octyl phenol ethoxylate) solution, a transposase solution, a tagmentation buffer, a cross-linking reversal solution, and amine- functionalized magnetic beads.
  • FIGS. 1A-1C High data quality from CUT&Tag-direct for whole cells.
  • 1A A comparison of H3K4me3 CUT&Tag tracks for K562 cells (tracks 2-6) at a representative 100-kb region of housekeeping genes, showing group-autoscaled profiles for 4 million mapped fragments from each sample.
  • 1B-1C Number of Peaks and Fraction of Reads in Peaks called using MACS2 on samples containing the indicated number of cells. Random samples of mapped fragments were drawn, mitochondrial reads were removed and MACS2 was used to call (narrow) peaks.
  • the number of peaks called for each sample is a measure of sensitivity, and the fraction of reads in peaks (FRiP, right) is a measure of specificity calculated for each sampling from 50,000 to 16 million fragments.
  • Nuclei data are from a previously described experiment (Example 1, Kaya-Okur HS, Janssens DH, Henikoff JG, Ahmad K, Henikoff S. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc. 2020;15(10):3264-83).
  • FIGS. 2A-2F High temperatures improve yield of small mouse fragments with FFPE-CUTAC.
  • 2A Scheme, where TL Prot K is Thermolabile Proteinase K (New England Biolabs). Created with BioRender.com. 2B) Arrhenius plot showing the recovery of fragments mapping to the Mm 10 build of the mouse genome as a function of temperature.
  • Deparaffinized FFPEs were scraped into cross-link reversal buffer (Example 1, Oba U, Kohashi K, Sangatsuda Y, Oda Y, Sonoda KH, Ohga S, et al. An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections.
  • H3K27ac 15 samples; 50:50 mixture of RNAPII-Ser5 and RNAPII-Ser2,5p: 14 samples. For each sample, mouse fragment lengths were divided by the total number of fragments before averaging. Lengths are plotted at single base-pair resolution. 2F) Average length distributions for on-slide samples grouped by cancer driver transgene (YAP1 : 12 samples; PDGFB: 7 samples; RELA: 12 samples) and Normal brain: 10 samples. Data are presented as mean values +/- SD in panels E-F. S
  • FIGS. 3A-3C Length distributions of DNAs tagmented by CUT&Tag of FFPEs.
  • 3A Lengths are plotted at single basepair resolution.
  • 3B Same as (A) except smoothed with a 5- bp window to iron out the 10-bp periodicity and facilitate comparisons.
  • 3C Same as (A) except for Mm 10 ChrM (mitochondrial) fragments from the same FFPEs as used for (A-B). The length distribution of MmlO ChrM fragments from mouse 3T3 cells is plotted for reference.
  • FIGS. 4A-4G Comparison of H3K27ac FFPE-CUTAC to FACT-seq and CUT&Tag of frozen unfixed samples.
  • (4A-4D) Representative examples of housekeeping gene regions were chosen to minimize the effect of cell-type differences between FFPE-CUTAC (three brain tumors) and FACT-seq (kidney).
  • Forebrain H3K27ac ChlP-seq and ATAC-seq samples from the ENCODE project are shown for comparison, using the same number of fragments (20 million) for each sample. Also shown are tracks from FFPE-CUTAC samples using an antibody to RNAPII-Ser2,5p.
  • FIGS. 5A-5D Volcano plots for pairwise comparisons between FFPE-CUTAC samples.
  • the Degust server (degust.erc.monash.edu/) was used with Voom/Limma defaults to generate volcano plots, where replicates consisted of a mix of samples run in parallel or on different days on FFPE slides from 8 different brain samples. (3 Normal, 3 YAP1, 1 PDGFB, 1 RELA). Input for each sample was 10-25% of an FFPE slide, which ranged from -50,000- 100,000 cells per 10-micron section.
  • FIGS. 6A-6L Top significant differences between tumor and normal and between tumors based on RNAPII-Ser5p FFPE-CUTAC comparisons.
  • 6F-6L Tracks centered around the cCRE for each of the strongest signals with FDRs ⁇ 0.05, ordered by increasing FDR (0.003 - 0.045).
  • FIGS. 7A-7B Comparisons between FFPE-CUTAC and RNA-seq. 7A) Scatterplots of representative FFPE-CUTAC replicate samples from RNAPII-Ser5p, RNAPII-Ser2,5p, RNAPII-Ser5p + RNAPII-Ser2,5p and H3K27ac. 7B) Examples of the best distinguished samples based on FDR. Pairwise comparisons between samples were used to choose examples in rank order based on FFPE-CUTAC FDR.
  • FIGS. 8A-8D FFPE-CUTAC distinguishes tumor from normal tissue within the same FFPE section.
  • RELA drives well-defined epidendymomas where dissection following tagmentation and transfer of whole sections to PCR tubes after RNAPII-Ser5p FFPE-CUTAC post-tagmentation successfully separated tumor from normal tissue with volcano plot results similar to that for RELA versus Normal brains (FIGS. 5A-5D).
  • 8B In contrast, PDGFB-driven gliomas are relatively diffuse, and separation of sections posttagmentation resulted in fewer significant target cCREs.
  • FIGS. 9A-9I FFPE-CUTAC produces high-quality data from liver FFPEs.
  • 9A-9D Representative tracks of liver tumor and normal liver FFPE-CUTAC and FACT-seq samples at the housekeeping gene regions depicted in FIGS. 4A-4D.
  • a track for Candidate cis- Regulatory Elements (cCREs) from the ENCODE project is shown above the data tracks, which are autoscaled for clarity.
  • 9E-9F Number of peaks and Fraction of Reads in Peaks (FRiP) called using MACS2 on samples containing the indicated number of cells for 7 liver tumor (magenta), 6 normal liver (blue) and 2 normal liver FACT-seq (green) samples.
  • 9G Cumulative logio plots of normalized counts intersecting cCREs versus logio rank for representative liver samples, where red marks dots with FDR ⁇ 0.05.
  • 9H Voom/Limma volcano plot for the 7 liver tumors versus 6 normal liver samples.
  • 91 Control volcano plot in which three liver tumor samples and 3 normal livers were exchanged for Voom/Limma analysis.
  • FIGS. 10A-10C Modified CUT&Tag-direct for whole cells and FFPEs.
  • 10A Scheme.
  • 10B Representative Tapestation profiles for whole-cell CUT&Tag-direct.
  • a log culture of K562 cells was supplemented with 10% DMSO, concentrated to 2 million cells/ml, aliquoted, slow-frozen in Mr. Frosty containers and stored at -80 °C. An aliquot was thawed and 15-60 pL was dispensed into PCR tubes for CUT&Tag-direct using an H3K27me3 antibody (CST cat. no. 9733).
  • 10C Tapestation profiles for FFPE CUTAC samples preincubated 85 °C 12 hr using four different antibodies on samples.
  • H3K27ac Abeam #4729.
  • a 10 pm section of a mouse brain tumor FFPE was deparaffinized using Option 1 (xylene). Note that both the CUTAC peaks the high-molecular weight smears scale with the amount of sample, likely representing ambient RNAs, which do not interfere with flow cell runs.
  • FIG. 11 Volcano plots of RNA-seq comparisons. Yapl : 3 replicates; Pdgfb: 4 replicates; RelA: 4 replicates; Naive: 7 replicates.
  • FIG. 12 Exemplary home workbench for CUT&Tag. Photo of example home workbench setup used for experiments in Example 1 protocol. A typical experiment begins by mixing cells with activated ConA beads in 32 single PCR tubes, with all liquid changes performed on the magnet stands. The only tube transfer is the removal of the purified sequencing-ready libraries from the SPRI beads to fresh tubes for Tapestation analysis and DNA sequencing.
  • FIG. 13 Image of part of an FFPE mouse brain tumor 10 pm shard after needle dispersion and 90 °C pre-treatments, stained with Trypan blue.
  • FIG. 14 Left: Reducing DNA contamination increases yields. Right: Arrhenius plot illustrates how high temperatures decrease the fraction of contaminating DNA, which when denatured is not a substrate for Tn5.
  • FIG. 15. Image of example transfer of paraffinized curls to mineral oil.
  • FIG. 16 Image of beads bound to tissue shards.
  • FIG. 17. Tapestation profiles for FFPE CUTAC samples pre-incubated 85 °C 12 hr using four different antibodies on samples. Each sample was divided 3/4- 1/4 in the TAPS- wash before fragment release. Antibodies (1 :25): RNAPII-Ser5p Cell Signaling Technology #13523, RNAPII-Ser2,5 Cell Signaling Technology #13546, H3K27ac: Abeam #4729. A 10 pm section of a mouse brain tumor FFPE was deparaffinized using Option 1 (xylene). Note that both the CUTAC peaks the high-molecular weight smears scale with the amount of sample. Use a 175-500 bp range for estimating molar concentration. There is no need to remove the high molecular weight smear, which is not tagmented and does not interfere with the flow cell run.
  • FIG. 18 A gene-rich housekeeping gene region was chosen to minimize the effect of cell-type differences between FFPE-CUTAC (A RelA-driven and two replicates of a PDGFB-driven brain tumor) and FACT-seq and CUT&Tag (kidney data from Zhao L, Xing P, Polavarapu VK, Zhao M, Valero-Martinez B, Dang Y, et al. FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers. Nucleic Acids Res. 2021;49(21):el25.).
  • a forebrain H3K27ac ChlP-seq sample from the ENCODE project is shown for comparison, using the same number of fragments (10 million) for each sample. Also shown are tracks from FFPE-CUTAC samples using an antibody to RNAPII- Ser2,5p. A track for Candidate cis-Regulatory Elements (cCREs) from the ENCODE project is shown above the data tracks, which are autoscaled for clarity.
  • cCREs Candidate cis-Regulatory Elements
  • FIG. 19 On-slide FFPE-CUTAC. Schematic of an example protocol.
  • FIG. 20 Image of a small slide holder that will hold two plastic film-wrapped slides without touching or disturbing the wrap. Closing the top will allow for long incubations without drying out. For small tissue sections (e.g., 1 cm 2 ), using small plastic wrap squares that cover the sample but do not wrap around the slide will require proportionally less volume, saving on reagent costs.
  • FIG. 21 Optional setup for incubating multiple slides with the same solution.
  • FIG. 22 Example of an incubation step.
  • On-slide FFPE-CUTAC was performed using a rabbit RNA Polymerase II Serine-5 monoclonal antibody (Cell Signaling Systems #13523).
  • Four slides from two mouse RELA transgene-driven ependymoma FFPE blocks (5 and 10 pm from the 33005 block and 10 pm from the 33003 block) were processed in parallel.
  • the slides were placed on top of plastic film over a black background for good visibility of tissue, slides were abutted and aligned for each incubation as indicated.
  • About 100 pl antibody or pAG-Tn5 solution was added dropwise to cover the tissue, and the plastic film was slowly pulled over the top edge, minimizing bubbles and wrinkles.
  • FIG. 23 Tapestation gel image of 1/10 th of each SPRI-bead purified DNA eluate from an on-slide experiment.
  • FIGS. 24A-24C Analyses of the data produced in an example CUTAC-FFPE experiment shown in Figures 20-21.
  • 24A Remainder of each (barcoded) sample was pooled together with other barcoded samples and sequenced on a NextSeq 2000 PE50 flow cell and the library size was estimated based on Picard Tools Mark Duplicates (68,089,523 in total) and plotted against the total number of reads (149,314,057 in total) for each sample. Total unique fragment estimates were: 10,582,472 (5 pm square), 20,708,800 (10 pm hexagon), 16,833,815 (5 pm pentagon) and 19,964,436 (10 pm triangle).
  • 24B Fragment length distributions of tumor and normal sections from all slides.
  • FIG. 25 Examples of moist chambers using wet paper towels in a plastic tray and staining dish. When covered slides stay wet under plastic wrap rectangles or squares (for small tissue sections and reduced volumes). Slides are placed in the rack for incubation, and afterwards are placed face up on the wet paper towel in the plastic tray to wash the bottom before removing the plastic wrap and rinsing the top.
  • FIG. 26 A curl (white) in a 1.5 mL Eppendorf tube.
  • FIGS. 27A-27C Analyses of the data produced in the experiment shown in Figures 20 and 21.
  • 27A Remainder of each (barcoded) sample was pooled together with other barcoded samples and sequenced on a NextSeq 2000 PE50 flow cell and the library size was estimated based on Picard Tools Mark Duplicates (68,089,523 in total) and plotted against the total number of reads (149,314,057 in total) for each sample. Total unique fragment estimates were: 10,582,472 (5 pm square), 20,708,800 (10 pm hexagon), 16,833,815 (5 pm pentagon) and 19,964,436 (10 pm triangle).
  • 27B Fragment length distributions of tumor and normal sections from all slides. Mean with standard deviation error bars.
  • FIGS. 28A-28F RNAPII-Ser5p FFPE-CUTAC directly maps hypertranscription.
  • 28A Model for hypertranscription in cancer: Paused RNAPII at active gene regulatory elements, such as promoters and enhancers, increases on average over the cell cycle resulting in a net proportional gain in RNAPII occupancy across the genome.
  • RNAPII FFPE- CUTAC hypertranscription genome-wide can be mapped using three complementary approaches: 1) Genome-scaled Tumor (T) minus Normal (N) counts at cCREs, 2) T - N at replication-coupled histone genes and 3) Sparse Enrichment Analysis for CUT&RUN (SEACR) Tumor peak calls using Normal as the background control.
  • T Genome-scaled Tumor
  • N Normal
  • SEACR Sparse Enrichment Analysis for CUT&RUN
  • 28B-28E Bland- Altman plots showing hypertranscription mapped over the 343,731 annotated mouse cCREs for tumor and normal sections dissected posttagmentation from a 10 micron FFPE slice from each of four different paraffin blocks.
  • Hypertranscription of a cCRE is defined as the excess of RNAPII-Ser5p in the indicated tumor over normal (Tumor minus Normal in normalized count units for MmlO-mapped fragments pooled from the same slide).
  • 28F Hypertranscription at replication-coupled histone genes. Slides used for PDGFB-2a-c were from the same paraffin block but used in different experiments, and all others were from different paraffin blocks.
  • FIGS. 29A-29I Hypertranscription in human Tumor-vs-Normal tissues. 29A-29H) All fragments were pooled from four slides from the same paraffin block and the number of fragments equalized between tumor and normal for each of the seven cancers. Bland-Altman plots showing hypertranscription mapped over the 984,834 annotated mouse cCREs for tumor and matched normal sections from 5 micron FFPE slices. Max Diffs displays the Tumor minus Normal maximum of the seven samples for each cCRE. 291) The minor human histone gene cluster on Chr 1 is shown, where tracks are autoscaled for each Tumor (red) and Normal (blue) pair. As individual samples are not intended to represent tumor types, sample names are abbreviations (FIG. 37).
  • FIGS. 30A-30D FFPE-CUTAC mitochondrial DNA signal is reduced in tumors.
  • 30B Same as (A) for RNAPII-Ser5p FFPE-CUTAC data for the seven human Tumor/Normal pairs used in this study.
  • 30C-30D ATAC-seq count data from The Cancer Genome Atlas (TCGA) (tumor) and ENCODE (normal) shows variability in ChrM percentages between tumors, consistent with our finding based on FFPE-CUTAC.
  • TCGA Cancer Genome Atlas
  • FIGS. 31A-31F Top-ranked human cCREs based on hypertranscription correspond to SEACR Tumor-vs-Normal RNAPII-Ser5p peaks.
  • tracks are shown for 50-kb regions around the #l-ranked cCRE based on Tumor (dark gray) and Normal (gray) counts.
  • Raw data tracks were group-autoscaled together for tumor (dark gray) and normal (gray), where SEACR Tumor peak calls (light gray) use Normal as the negative control.
  • Gene annotations and cCREs black rectangles are shown at top.
  • FIGS. 32A-32F Hypertranscription differs between human liver tumors.
  • 32A-32D Top-ranked cCREs based on liver tumors 1 and 2 (dark gray) and matched normal (gray) counts. Tumor/Normal tracks and Tumors 3-5 are group-autoscaled.
  • 32E Same as (A), except for the minor histone gene cluster on Chromosome 1.
  • 32F Levels of hypertranscription differ between different hepatocarcinomas (Tumor 1 : solid lines, Tumor 2 dotted lines, where tumor is dark gray and normal is gray).
  • FIG. 33 Tight clustering of tumor samples. UMAP of 114 human tumor samples (upper panel). Lower panel, Same as upper panel except shaded for sequencing depth and indicating homogeneous tumor clusters.
  • FIGS. 34A-34F Hypertranscription identifies likely HER2 amplifications and regions of linkage disequilibrium.
  • 34A Raw data tracks for the 1-Mb region on Chromosome 17q21 were group-autoscaled together for tumor and normal, where SEACR Tumor peak calls use Normal as the negative control. Broad regions of prominent hypertranscription, indicate likely HER2 amplifications in both tumors.
  • 34B Raw data tracks for the 250-kb 17ql2 region amplified in Br but evidently not in Co.
  • 34C Raw data tracks for the CCNK promoter region, where the normalized count increase in the Br tumor relative to normal over the 10-kb region shown is 5.4-fold and for Co is 2.1 -fold and the range for the other five tumors is 0.9-2.5.
  • 34D-34E The two 1-Mb regions displayed in (C-D) were tiled with 1-kb bins and count density curves were fitted for all 7 tumor-normal pairs. Arrows mark the locations of indicated promoter peaks in the breast and colon tumors.
  • 34F Individual broad summits in (D-E) were zoomed-in and rescaled on x-axis centered over the indicated promoter peak and superimposed over raw normalized count tracks scaled to the height of the central peak.
  • FIGS. 35A-35H RNAPII-Ser5p FFPE-CUTAC shows stronger and more frequent changes in up-regulation than down-regulation of cCREs.
  • the Voom/Limma option of the Degust server (degust.erc.monash.edu/) was applied to mouse cCRE RNAPII-Ser5p FFPE-CUTAC data from pooled replicates from 5 RELA and 4 PDGFB experiments.
  • Normalized counts are the fraction of counts at each base pair scaled by the size of the MmlO reference sequence (2,818,974,548), so that if the counts are uniformly distributed across the reference sequence there would be one at each position.
  • 35A-35B Both RELA and PDGFB tumor sections show higher counts than normal sections but significant RELA changes both up and down are far stronger than PDGFB changes, confirmed in a head-to-head comparison between tumors and normal sections.
  • 35C-35E Same as (A-B) except using either RNAPII-SerSp or histone H3K27ac antibodies for FFPE-CUTAC and using entire 10 pm curls divided into 4-8 samples per curl for PCR and sequencing.
  • MA plots data were merged from multiple experiments and equalized by downsampling to 10 million fragments, with 4 merged replicates per sample. DAP-stained slides for each paraffin block used, with the total fraction of tumor indicated in parentheses. (35F-35H) Voom/Limma was used to construct MA plots based on individual 10 pm sections from single slides corresponding to the boxed sections on slides DAP-stained for tumor-driver transgene expression. Numbers in parentheses are percentages of tumor cells based on numbers of stained and unstained cells within the boxed sections.
  • FIG. 36 Hypertranscription mapped over the 343,731 ENCODE-annotated mouse cCREs categorized by regulatory element type.
  • Figure 28 For each tumor and normal sample, we counted the number of mapped fragments spanning each base-pair in a cCRE scaled to the mouse genome and averaged the number of counts over that cCRE.
  • FIG. 37 Photographs of 5 pm FFPE sections from human tumor and adjacent normal tissues.
  • Pathology classification, age and sex were provided by the vendor (BioChain). Each image spans the width of a standard charged microscope slide, where the tissue is visible under the paraffin skin.
  • On-slide RNAPII-Ser5p FFPE-CUTAC was applied to slides in parallel, using a total of four slides each for 100 separate samples in all to produce the data analyzed in this study. To avoid the impression that these individual tumors are representative of their tumor types, their designations are abbreviated: Br, Co, Ki, Li, Lu, Re and St.
  • FIGS. 38A-38X Hypertranscription in human Tumor-vs-Normal tissues. Related to Figure 29. 38A-38H) Same data as in Figure 29A-29H, except plotted as in Figure 37 to facilitate comparisons. 38I-38P) Combined data from a single slide with duplicate removal. 38Q-38X) Combined data from 4 slides after removing duplicates and equalizing the number of fragments between tumor and normal sections. Number of unique fragments per sample in each Tumor/Normal pair: Br: 1,125,608; Co: 3,712,097; Ki: 2,031,893; Li: 2,983,411; Lu: 1,123,638; Re: 3,284,736; St: 719,598. [0063] FIGS.
  • 39A-39L Focal hypertranscribed regulatory elements embedded in broad regions of hypertranscription on Chromosome 17ql2-22.
  • 39A-39F The six most highly transcribed cCREs within the ⁇ 5 Mb region of Chromosome 17ql.2-2.2 are displayed with each tumor (dark gray) and normal (gray) pair scaled to one another so that peaks can be observed in all samples.
  • SEACR peaks (light gray) are group-autoscaled in all panels.
  • FIGS. 40A-40E Weak RNAPII upregulation of RNAPII of the top-ranked loci outside of the HER2 amplicon.
  • Figure 34C-34D See Figure 34C-34D for details regarding top-ranked loci outside of the HER2 amplicon.
  • Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • the term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and/or C- terminal ends of the recited sequence or additional nucleotides on the 5’ and/or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered.
  • the total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together.
  • the term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities/properties (e.g., remodeling activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleotides
  • plasmids vectors
  • genomic DNA
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.
  • modified nucleotides such as methylated nucleotides and nucleotide analogs or derivatives (e.g, inosine or phosphorothioate nucleotides).
  • nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.
  • modulate refers to enhancement (e.g, an increase) or inhibition (e.g., a decrease) in the specified level or activity.
  • the term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold and/or can be expressed in the enhancement and/or increase of a specified level and/or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.
  • inhibit or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).
  • contact or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.
  • DNA Integrity Number or RNA Integrity Number (RIN) refers to a numerical value quote as a measure of the quality of the DNA or RNA.
  • a DIN/RIN can be measured using DNA/RNA quantification machines, for example, by the Agilent Tapestation® or Bioanalyzer.
  • a DIN/RIN value ranging between 1 and 10 can be assigned to the DNA/RNA, with 10 being completely intact material and 1 being completely degraded.
  • the DIN score for an FFPE sample is evaluated subsequent to deparaffinazation of the sample, and may comprise extracting cells, nuclei, or DNA isolated from the sample.
  • the high sensitivity of the present methods allows evaluation of samples with a DIN score of at least 5, 4.5, 4, 3.5, 3, 2.5, or 2.
  • Previous CUT&Tag-based methods show limited compatibility with analysis of FFPE samples.
  • the present invention relates to the use (and improvements to) CUTAC to enable high-throughput FFPE tissue analysis.
  • CUTAC methods are described, for example, in International Patent Publication WO 2022/056309, incorporated herein by reference in its entirety. Applicants herein leverage the high sensitivity of CUTAC along with further revisions to those methods to get high signal to noise in FFPE samples, including highly degraded FFPE samples.
  • the CUTAC workflow produces ⁇ 120-bp fragments that not only increases mapping resolution and sensitivity, but also helps overcome DNA degradation caused by fixation and cross-linking by increasing the likelihood of two successful tagmentation events occurring on an intact segment of DNA.
  • the formaldehyde treatment in a FFPE sample forms covalent bonds between DNA and lysine-rich histones in nucleosomes rendering them inflexible, so that open chromatin gaps are the accessible DNA in the nucleus.
  • the presently disclosed methods take advantage of the hyperaccessibility and abundance of the targeted epitope and the impermeability of lysine-rich histone cross-linked chromatin to achieve exceptional signal-to-noise from FFPE samples.
  • the disclosed methods can use RNAPII to map the transcriptional machinery itself directly on the DNA regulatory elements, such that direct measurements of transcription initiation are obtained that can characterize hypertranscription at active regulatory elements genome-wide, rather than inferences based on estimating steady-state mRNA abundances.
  • the present invention is related to methods for measuring hypertranscription by quantifying incremental increases or decreases in RNAPII over hundreds of thousands of loci, allowing high resolution results while using low sequencing depth without reference to external information and allowing detection of genome-wide hypertranscription.
  • the methods of measuring hypertranscription disclosed herein allow identification of loci amplifications and probable clonal selection events without relying on reference to any external data.
  • FFPE- CUTAC methods described herein can be utilized with automation to allow for routine cancer screening and other personalized medicine applications.
  • the methods can be performed rapidly at low-cost ( ⁇ $50 per sample) providing value as a general clinical diagnostic and research tool.
  • an in situ method of mapping the location of a protein on chromatin in a cell from a FFPE sample comprising the steps of treating the FFPE sample to remove the paraffin; permeabilizing the sample; contacting the sample with a first affinity reagent that specifically binds to a chromatin protein, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules; excising the tagged DNA segment associated with the chromatin protein; and determining the nucleotide sequence of the excised tagged DNA segment, thereby mapping the genomic location of the targeted protein on chromatin.
  • a DNA-based in situ method for measuring transcription in a cell from a FFPE sample comprising: treating the FFPE sample to remove the paraffin; permeabilizing the sample; contacting the sample with a first affinity reagent that specifically binds to a protein involved in transcription regulation, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules; excising the tagged DNA segment associated with the protein involved in transcription regulation; and determining the nucleotide sequence of the excised tagged DNA segment, thereby mapping transcriptional activity on chromatin.
  • treating the FFPE sample to remove the paraffin can comprise applying high heat to the sample.
  • high heat can include heating a sample above 50°C, which may be at least 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 90°C.
  • removing paraffin comprises incubating the sample at least at 75°C, 80°C, 85°C, or 90°C.
  • the sample may be heated for between about 5 minutes and 3 or more hours (See, e.g., Example 3, step 25 and accompanying note describing extension of incubation times), which may be dependent at least in part on the sample type, for example, whether sample is tissue on a slide, or cells or nuclei associate with beads, or samples in nanowells.
  • removing paraffin comprises heating at 85-90°C for between 1 hour and 16 hours.
  • the method comprises isolating nuclei with heat and minimal mechanical processing.
  • the method comprises isolating nuclei without (i.e., is devoid of) enzymatic processing of the tissue for isolating nuclei.
  • the sample is further treated with cross-link reversal buffer, which may comprise Tri s(hydroxymethyl)aminom ethane hydrochloride (Tris HC1) and/or Ethylenediaminetetraacetic acid (EDTA).
  • cross-link reversal buffer which may comprise Tri s(hydroxymethyl)aminom ethane hydrochloride (Tris HC1) and/or Ethylenediaminetetraacetic acid (EDTA).
  • a cell (or nucleus) in the sample is permeabilized with a detergent, e.g., by digitonin.
  • a cell and/or nucleus of the cell in the sample is permeabilized by the step of removing the paraffin with heat in the cross-link reversal buffer.
  • the addition of Triton®- XI 00 to buffer solutions used in several steps of the methods helps maintain cell permeability.
  • the method comprises separating the sample into tissue fragments, cells, or nuclei before or after the step of permeabilizing the sample.
  • the sample may comprise a tissue sample, e.g., a curl, a slice, a punch, or other FFPE tissue sample.
  • the sample can comprise about 1,000 cells to about 2,000,000 cells or more, or any range therein.
  • the sample is separated into single cells and/or nuclei prior to contacting the sample with the first affinity reagent.
  • the method is performed on fragments of a sample that has been mechanically digested.
  • Example 3 An example embodiment of mechanical separation is provided in Example 3 describing the mortar and pestle protocol.
  • the sample is sheared.
  • methods can comprise obtaining tissue from a slide, for example, by optionally dicing or otherwise sectioning the tissue sample on the slide and scraping the tissue from the slide and further forcing a solution comprising the tissue sample multiple times through a needle (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times) to thereby provide fragments of a sample.
  • a needle e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times
  • the method can be performed on a FFPE curl.
  • the step of treating the FFPE curl sample comprises adding mineral oil to the sample and heating the sampled at 85-90°C for between 3-10 minutes (e.g., 5 minutes) to melt paraffin, and homogenizing the sample with a pestle.
  • the method can comprise adding cross-link reversal buffer comprising Tris- HC1 at a pH between about 7.5 and 9.0 (e.g., pH8.0) and amine functionalized paramagnetic beads in a ratio of, for example 1 : 10.
  • homogenization can be repeated followed by a subsequent addition of crosslink reversal buffer.
  • the cross-link buffer utilized is warmed prior to addition.
  • the samples are incubated at 85-90°C for between 1 hour-14 hours followed by vortexing, centrifuging, and removing the mineral oil.
  • Mineral oil can then be added, mixed by inversion, centrifuged and subsequently removed with the exception of a thin oil layer at the top of the sample.
  • the method can further comprise adding paramagnetic beads, for example, agarose glutathione paramagnetic beads, and mixing the samples.
  • the method can comprise exposing the samples comprising the paramagnetic beads to a strong magnet, followed by removing the supernatant, and re-suspending the remaining bead-bound homogenate in a buffer comprising Triton® X-100 and optionally HEPES pH 7.5, NaCl, spermidine, EDTA, and/or EDTA-free protease inhibitor prior to adding a first affinity reagent.
  • a buffer comprising Triton® X-100 and optionally HEPES pH 7.5, NaCl, spermidine, EDTA, and/or EDTA-free protease inhibitor prior to adding a first affinity reagent.
  • less than 10% of a curl for example, 5% of a curl, is sufficient for generating a single library using the methods described herein.
  • the method is performed on a solid support, for example a bead, a slide, a well (e.g., a microwell or nanowell) and/or the wall of a microtiter plate.
  • the bead may be an amine-functionalized bead, for example, an agarose-glutathione bead or a lectin-coated bead (e.g., Concanavalin A).
  • the bead is a magnetic bead.
  • the method is performed directly on a slide comprising the sample, e.g., a tissue sample.
  • the method performed on a slide produces spatially resolved results, as described further herein.
  • the method further comprises tagging each of a plurality of cells with a cell specific barcode or combination of barcodes unique to a location in a three-dimensional plurality of cells. Labeling can comprise inserting barcodes via transposase transposition or other ligation techniques (e.g., splint ligation) that can be followed by high-throughput sequencing to thereby allow spatial-resolution genome-wide mapping of chromatin protein or a protein involved in transcription regulation in tissue at a cellular level.
  • the method can further comprise the step of imaging the three-dimensional plurality of cells prior to the step of excising the tagged DNA.
  • H4C / IF imaging could be used to register histology information to spatial sequencing data.
  • integrating cell morphology information with spatial epigenomic mapping may provide deeper insights into how tissues change due to aging, injury, disease and/or treatment.
  • cells comprising tags e.g., DNA barcodes, for example fluorescently labelled DNA oligomers, are imaged to thereby correlate the cell and its corresponding DNA barcodes to allow for identification of tracking of the cell location.
  • the methods comprise using contaminating bacterial DNA as a calibration standard to normalize samples.
  • the contaminating bacterial DNA is Rhodococcus DNA.
  • FFPE samples may be contaminated with the gram-positive bacterium Rhodococcus erythropolis and utilizing Rhodococcus DNA as the calibrating may avoid challenges when using spike-in controls with a FFPE sample.
  • the method can comprise using Rhodococcus DNA and/or nucleosome-based spike-ins.
  • methods comprise using nucleosome-based spike-ins (e.g. containing histone PTMs or other epitopes in chromatin associated protein) as previously described in, for example, International Patent Publication Nos. WO 2015117145, WO 2013184930, WO 2020132388, and WO 2020168151.
  • Methods may further comprise the step of deproteinating the DNA segment with an enzyme, e.g., a proteinase.
  • the method comprises treating the sample with a serine protease, e.g., proteinase K, prior to excising the tagged DNA segment.
  • the proteinase K can be provided in a solution comprising SDS.
  • the SDS may be used at greater than 0.5%, for example, greater than 0.6%, 0.7%. 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%.
  • the method comprises contacting the tagged chromatin DNA segments with an SDS solution comprising proteinase K, for example 5%, 4%, 3%, 2%, 1%, 0.75%, or 0.5% SDS.
  • excising the tagged DNA can be performed using heat, for example, at a temperature of at least 35 °C, 40°C, 45 °C, or 50 °C.
  • the SDS is supplemented with a 1 : 10 proteinase K to a solution used for fragment release.
  • the tagged DNA segments are contacted at 30°C to 45°C (e.g., 37°C) for between 0.5 hours-2.5 hours (e.g., 1 hour), followed by 50°C to 65°C (e.g., 58°C) for 0.5 hours-2.5 hours (e.g., 1 hour).
  • the step can be quenched by adding a solution of Triton®-X100, for example, 1% to 12% Triton®-X100 (e.g., 6%).
  • the proteinase K can be a therm olabile proteinase K, which was cloned from Engyodontium album (formerly Tritirachium album) and mutagenized to increase thermolability of the enzyme available from New England Biolabs.
  • a supernatant containing the cleaved segments is optionally treated with a proteinase, and DNA is quantified, for example, with imaging of stained DNA of the cleaved segments.
  • the sample is contacted with a first affinity reagent that specifically binds to a chromatin protein or a protein involved in transcription regulation.
  • a protein involved in transcription regulation can include proteins that localize near accessible (or “open) chromatin (e.g., H3K4me2 or RNAPIIS5p).
  • an affinity reagent to a phosphoform of the C-terminal domain of RNAPII can be used in the methods described herein.
  • the initiation form of RNAPII which has a serine-5 phosphate on the repeated heptameric C terminal domain of the largest subunit (referred to as RNAPIIS5P), precisely aligns with transcription-coupled chromatin accessibility.
  • RNAPII which has a serine-2 phosphate on the repeated heptameric C-terminal domain of the largest subunit (referred to as RNAPIIS2P), also precisely aligns with transcription- coupled chromatin accessibility.
  • Example phosphoforms of the C-terminal domain of RNAPII include RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII- Ser2/5, or RNAPII- Ser5/7 and an affinity reagent such as an antibody that specifically binds RNAPII can be utilized for assays related to transcription regulation and/or chromatin accessibility.
  • Affinity reagents for chromatin proteins include, but are not limited to, for example, reagents that specifically bind to markers for negative regulatory elements (e.g., H3K27me3 or H3K9me3).
  • the sample is contacted with a first affinity reagent that specifically binds to a targeted chromatin protein or a protein involved in transcription regulation.
  • a first affinity reagent that specifically binds to a targeted chromatin protein or a protein involved in transcription regulation.
  • the formaldehyde treatment of a FFPE sample forms covalent bonds between DNA and lysine-rich histones in nucleosomes rendering them inflexible, so that open chromatin gaps are the accessible DNA in the nucleus.
  • the presently disclosed methods can take advantage of the hyperaccessibility and abundance of the targeted epitope and the impermeability of histone cross-linked chromatin to achieve exceptional signal-to-noise.
  • the first affinity reagent is directly coupled to at least one transposase.
  • the at least one transposase comprises a Tn5 transposase.
  • the first affinity reagent and transposase are disposed in a fusion protein.
  • the first affinity reagent is indirectly coupled to the at least one transposase.
  • the transposase is linked to a specific binding agent that specifically binds the first affinity reagent.
  • the first affinity reagent is bound by a second affinity reagent.
  • the method further comprises contacting the cell with a second affinity reagent that specifically binds the first affinity reagent, and wherein the transposase is linked to a specific binding agent that specifically binds the second affinity reagent.
  • the method further comprises contacting the cell with a second affinity reagent that specifically binds the first affinity reagent, contacting the cell with a third affinity reagent that specifically binds the second affinity reagent, and wherein the transposase is linked to a specific binding agent that specifically binds the third affinity reagent.
  • a second affinity reagent is bound by a third affinity reagent.
  • the first, second, or third affinity reagent is directly coupled to the at least one transposome.
  • the first, second, or third affinity reagent is indirectly coupled to the at least one transposome.
  • the transposome comprises a fusion protein of the transposase and the binding agent.
  • the transposome can comprise a Tn5 transposase domain and protein A or a binding domain thereof, protein G or a binding domain thereof, or a protein A/G hybrid binding domain.
  • the first, second, and/or third affinity reagents independently is or comprises an antibody, an antibody-like molecule, a DARPin, an aptamer, a chromatinbinding protein, other specific binding molecule, or a functional antigen-binding domain thereof.
  • the antibody-like molecule is an antibody fragment and/or antibody derivative.
  • the antibody-like molecule is a single chain antibody, a bispecific antibody, an Fab fragment, an F(ab)2 fragment, a VHH fragment, a VNAR fragment, or a nanobody.
  • the single-chain antibody is a single chain variable fragment (scFv), or a single-chain Fab fragment (scFab).
  • the first, second, and/or third affinity reagent is an antibody to a phosphoform of the C-terminal domain of RNA polymerase II (RNAPII), such as RNAPII-Ser2, RNAPII- Ser5, RNAPII-Ser7, RNAPII- Ser2/5, or RNAPII-Ser5/7.
  • RNAPII RNA polymerase II
  • Methods can comprise activating at least one transposase under low ionic conditions.
  • the use of low-salt tagmentation after stringent washes allows for tight binding of the Tn5 transposome and allows for epitopes flanking promoters and enhancers, such as RNAPII epitopes, to release subnucleosomal fragments preferentially, where tagmentation occurs within gaps in the chromatin landscape where these epitopes are located.
  • low ionic conditions comprise an ionic concentration of less than 10 mM.
  • activating the at least one transposase under low ionic conditions can comprise contacting the transposase with a sufficient amount of Mg ++ (such as in the salt form of MgC12 or MgSCh), for example, from about 0.1 mM Mg ++ to about 10 mM Mg ++ .
  • the low ionic conditions comprise a solution of MgCh and/or TAPS buffer, for example, MgCh at lOmM or less and/or TAPS buffer at 5 mM or less.
  • activating the at least one transposase under low ionic conditions is characterized by low monovalent ionic concentration of less than about 10 mM, for example, between about 1 mM to about 10 mM, about 2 mM to about 9 mM, about 3 mM to about 8 mM, about 4 mM to about 7 mM, about 5 mM to about 6mM, or any range therein.
  • the salt component of the reaction environment is NaCl, but other sources of monovalent ions are possible.
  • the monovalent ions can be supplied by salts with monovalent cations such as Na+, Li+, etc., or anions such as C1-.
  • the low ionic conditions can further comprise 1,6-hexanediol, a strongly polar aliphatic alcohol, and/or 10% dimethylformamide, a strongly polar amide.
  • 1,6-hexanediol a strongly polar aliphatic alcohol
  • dimethylformamide a strongly polar amide
  • the step of contacting the permeabilized cell with the first affinity reagent and/or the step of activating the at least one transposase and tagging the chromatin DNA are performed with a buffer comprising Triton® X-100 (octyl phenol ethoxylate).
  • Triton® X-100 is provided in a buffer at 20% by weight or less, for example, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, , 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03% or 0.02% or less.
  • the buffer comprising Triton® X-100 is provided in a ratio in solutions (e.g., transposases solution, primary or secondary affinity reagent (antibody) solution) at 1 : 15 - 1 :30, for example 1 :20 or 1 :25, solutiombuffer comprising Triton® X-100.
  • solutions e.g., transposases solution, primary or secondary affinity reagent (antibody) solution
  • solutiombuffer comprising Triton® X-100.
  • one or more of the steps of contacting the sample with a first affinity reagent which may comprise incubation, binding of a transposome, and activating at least one transposase to thereby cleave and tag DNA (e.g., tagmentation) utilize buffers comprising Triton®-X100, for example, 0.05% Triton®-X100.
  • a first affinity reagent e.g., antibody
  • activating at least one transposase to thereby cleave and tag DNA e.g., tagmentation
  • buffers comprising Triton®-X100, for example, 0.05% Triton®-X100.
  • Triton®-X100 for example, 0.05% Triton®-X100.
  • a solution comprising Triton®-X100 can be used to quench the reaction, i.e., sequesters SDS in micelles.
  • the method can be performed on whole cells without the need to purify nuclei.
  • the tagged DNA segment that is excised from chromatin is isolated by capturing supernatant in which the tagged DNA segment is released.
  • the excised chromatin DNA fragments are purified by immobilizing the fragments on a solid support, such as a bead, membrane, or surface (e.g,. a well or tube) that is coated with an affinity molecule suitable for immobilizing the excised chromatin DNA.
  • the affinity molecule is silica or magnetic beads (SPRI beads).
  • a library e.g., for next generation sequencing applications, such as Illumina® sequencing (Illumina® Inc., San Diego, CA) is constructed on magnetic particles.
  • the same DNA absorbing magnetic beads can then be used to purify the resulting library.
  • the excised chromatin DNA are purified after they have been released from the specific chromatin-associated factor and or antibody with which or to which the nucleic acid fragments were bound.
  • the methods yield ⁇ 120-bp fragments (e.g., 115 bp, 110 bp, 100 bp, 95 bp, 90 bp or less) released which is relatively robust to the serious DNA degradation that occurs during cross-link reversal.
  • the method comprises performing PCR.
  • PCR is performed with an extension step, for example, 10 sec 98°C denaturation, 30 sec 63 °C annealing and 1 min 72°C extension for 10-14 cycles, for example, 12 or 13 cycles.
  • the isolated tagged DNA segment that is excised from the chromatin can be subject to further analysis, such as size characterization, or full sequencing.
  • a further advantage of providing an affinity surface in a well or as a bead, e.g., magnetic beads is that the disclosed methods may be adapted for parallel processing of multiple samples, such as in a 96-well format or microfluidic platform, from starting chromatin material to the end of a sequencing library construction and purification.
  • the methods herein employing CUTAC can be used in conjunction with spatial analysis. For example, using in situ methods on the FFPE sample can be performed on the sample directly on a slide and then subjected to spatial analysis.
  • the methods herein can comprise isolating nuclei from a FFPE sample prior to performing an assay as described herein, followed by single cell (SC) approaches.
  • SC single cell
  • SC CUT&Tag first using established SC platforms, including the ICELL8 platform (Kaya-Okur, Wu et al. 2019) and the Chromium platform from lOx Genomics (Wu, Furlan et al. 2021).
  • SCs e.g., scChIC-seq (Ku, Nakamura et al. 2019), CoBATCH (Wang, Xiong et al. 2019), scCUT&RUN (Hainer, Boskovic et al. 2019)).
  • multiomic CUT&Tag e.g., Paired- Tag (Zhu, Zhang et al. 2021), scCUT&Tag-Pro (Zhang, Srivastava et al. 2021) have been developed.
  • Such approaches can be adapted with the disclosure herein for use with the described methods.
  • the transposome comprises a nucleotide barcode sequence.
  • Barcode identifier sequences are known in the art and typically comprise about 6 to 25 nucleotides in length.
  • the barcode sequence and methods of incorporation and use can be as described in International Patent Publication No. WO 2019140082 and International Patent Publication No. WO 2020132388, incorporated herein by reference.
  • Barcoding can alternatively or additionally be incorporated via other ligation strategies, including, for example, splint ligation or sticky ligation, with methods including split-and-pool barcoding. See, e.g., Satz, A.L., Brunschweiger, A., Flanagan, M.E. et al. DNA-encoded chemical libraries.
  • the method can further comprise evaluating a DNA Integrity Number (DIN) value for the sample e.g., after the isolation of excised tagged DNA from the sample.
  • DIN DNA Integrity Number
  • a portion of a sample is utilized for evaluating DIN value subsequent to removal of paraffin, with the remainder of the sample being used in the methods described herein.
  • one or more steps of the methods of the invention are carried out only when the DIN is greater than or equal to 3. See, e.g., Chougule et al., Comprehensive Development and Implementation of Good Laboratory Practice for NGS Based Targeted Panel on Solid Tumor FFPE Tissues in Diagnostics, Diagnostics, 2022, 12, 1291; doi: 103390/diagnostics 12051291.
  • an amount of DNA evaluated in the methods is measured after isolating the DNA fragments.
  • the DNA can be detected by the addition of nucleic acid stains, such as intercalating dyes (e.g., ethidium bromide and propidium iodide, SYBRTM Gold, SYBRTM Green I and SYBRTM Green II, cyanine based dyes), minor groove binders (e.g., DAPI, Hoechst, TOTO-1, indoles, imidazoles, and PicoGreenTM) and other stains (e.g., acridine orange, 7-AAD, hydroxystilbamidine (H22845), and LDS 751). Stains may be selected based on desired detection methods.
  • Quantifying DNA can comprise contacting the cleaved or excised fragment with a nucleic acid stain.
  • the methods may comprise quantifying DNA by methods such as spectrophotometry.
  • the methods described herein can comprise identifying transcriptional activity or mapping the location of a protein on chromatin that is indicative of a disease or disorder.
  • the methods described herein can further comprise detecting the amount of mtDNA in a sample, which can further indicate presence of a disease or disorder.
  • a method of monitoring a disease or disorder comprising performing a method as described herein from samples obtained at two or more points in time from the same subject, and comparing an amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin in each sample to a reference and/or to each other.
  • the amount of protein or transcription can be indicative of worsening (e.g., increased disease) or improving disease (lessening of the disease).
  • the reference control may be an aggregate of normal or healthy patients, e.g., one or more patients without the disease. Such reference controls can include healthy population of a particular age, gender, race or other variable.
  • the reference control comprises comparing a diseased sample to a normal sample from the subject, for example, matched tumor and normal tissue.
  • diseased tissue and normal tissue are derived from the same tissue sample, e.g., from the same section or different sections.
  • a method of monitoring a disease or disorder comprises determining efficacy of a treatment.
  • the method comprising performing a method as described herein from samples obtained at two or more points in time from the same subject receiving the treatment and comparing an amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin in each sample to a reference and/or to each other.
  • determining efficacy of a treatment comprises measuring the amount of protein or transcription is indicative of worsening (e.g., increased disease) or improving disease (lessening of the disease) as thereby indicative of efficacy of the treatment.
  • the differences in the amount and/or genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin at the two or more points in time indicate efficacy of a treatment of the disease or disorder in the subject.
  • the method can monitor disease progression and/or make treatment decisions for subjects based on changes in the amount and/or genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin.
  • the presently described methods can be used for detection and analysis of amplifications and clonal selection during cancer progression and therapeutic treatment. See, Example 4.
  • the reference control may be an aggregate of normal or healthy patients, e.g., one or more patients without the disease.
  • Such reference controls can include healthy population of a particular age, gender, race or other variable.
  • the reference control an also comprise healthy tissue from the subject and/or the sample comprising diseased tissue (e.g, tumor).
  • the first sample is obtained from a subject prior to beginning of treatment.
  • the second sample is obtained during and/or after treatment.
  • a method of diagnosing a disease or disorder in a subject comprising performing a method as described herein on a sample from the subject, and diagnosing the subject as having the disease or disorder based on an amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin to thereby diagnose the subject as having the disease or disorder.
  • the methods comprise correlating the interactions of a target nucleic acid with proteins and/or nucleic acid with a disease state, for example cancer, or an infection, such as a viral or bacterial infection.
  • the profile of the targeted protein on chromatin and/or the transcriptional activity on chromatin can be used to identify binding proteins and/or nucleic acids that are relevant in a disease state such as cancer, for example to identify particular proteins and/or nucleic acids as potential diagnostic and/or therapeutic targets.
  • the method can comprise diagnosing a subject with cancer based on the amount and/or the genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin which can comprise one or more genes in Table 2. [0113]
  • the methods described herein can further comprise comparing the amount and/or genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin with a control reference.
  • the reference control comprises comparing a diseased sample to a normal sample from the subject, for example, matched tumor and normal tissue.
  • diseased tissue and normal tissue are derived from the same tissue sample, e.g., from the same section or different sections.
  • a method of prognosing a disease or disorder in a subject comprising performing a method as described herein on a sample from the subject, and prognosing the disease or disorder in the subject based on the amount and/or genomic location of the targeted protein on chromatin and/or the transcriptional activity on chromatin.
  • a protein involved in transcription regulation can include proteins for chromatin accessibility (e.g., H3K4me2 or RNAPIIS5p).
  • Example phosphoforms of the C-terminal domain of RNAPII that can be used include RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2/5, or RNAPII-Ser5/7.
  • Chromatin-associated factors are factors that can be found at one or more sites on the chromatin and/or that may associate with chromatin in a transient manner.
  • low abundance chromatin-associated factors include, but are not limited to, transcription factors (e.g. , tumor suppressors, oncogenes, cell cycle regulators, development and/or differentiation factors, general transcription factors (TFs)), ATP-dependent chromatin remodelers (e.g., (P)BAF, M0T1, ISWI, INO80, CHD1), activator (e.g. , histone acetyl transferase (HAT)) complexes, repressor (e.g.
  • transcription factors e.g. , tumor suppressors, oncogenes, cell cycle regulators, development and/or differentiation factors, general transcription factors (TFs)
  • ATP-dependent chromatin remodelers e.g., (P)BAF, M0T1, ISWI, INO80, CHD1
  • activator
  • histone deacetylase (HD AC)) complexes e.g., histone (de-) methylases, DNA methylases, replication factors and the like.
  • factors may interact with the chromatin (DNA, histones) at particular phases of the cell cycle (e.g., Gl, S, G2, M- phase), upon certain environmental cues (e.g., growth and other stimulating signals, DNA damage signals, cell death signals) upon transfection and transient or stable expression (e.g., recombinant factors) or upon infection (e.g., viral factors).
  • Histones may be modified at histone tails through posttranslational modifications which alter their interaction with DNA and nuclear proteins and influence for example gene regulation, DNA repair and chromosome condensation.
  • the H3 and H4 histones have long tails protruding from the nucleosome which can be covalently modified, for example by methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination and ADP-ribosylation.
  • the core of the histones H2A and H2B can also be modified.
  • Example chromatin proteins include, but are not limited to, methylated H3K, such as H3K4me2 or H3K4me3, methylated H3K, such as H3K27me3, and acetylated H3K27 (H3K27ac) chromatin proteins.
  • the disclosed methods can be used for monitoring disease states, such as disease state in an organism, for example a plant or an animal subject, such as a mammalian subject, for example a human subject.
  • Certain disease states may be caused and/or characterized by differential binding of proteins and/or nucleic acids to chromatin DNA in vivo. For example, certain interactions may occur in a diseased cell but not in a normal cell.
  • a profile of the interaction can be generated allowing correlation with a disease state.
  • an interaction profile for a particular disease or disorder state e.g., infection, cancer, autoimmune disorder
  • an interaction profile for a particular subject, subpopulation or population can be generated using the methods described herein that can be used for diagnosis or prognosis of subjects with a similar interaction profile.
  • aspects of the disclosed methods relate to correlating the interactions of a target nucleic acid with proteins and/or nucleic acid with a disease state, for example cancer, or an infection, such as a viral or bacterial infection.
  • a method of detecting hypertranscription in a sample comprising performing a method as described herein, wherein an increased amount of transcriptional activity on chromatin thereby detects hypertranscription in the sample.
  • the method may comprise direct measurements of transcription initiation, elongation, and termination by mapping and quantitating RNAPII to thereby characterize hypertranscription at active regulatory elements.
  • the method comprises detecting RNAPII at non-coding regions, including, for example, enhancers (e.g., proxy enhancer activation.
  • Hypertranscription refers to a global increase in nascent transcription and can be measured across the genome, mapping hypertranscription at regulatory elements across the genome.
  • hypertranscription can be quantified, which can comprise normalizing count differences between tumor tissue sample and normal tissue sample from the same subject and/or same FFPE sample, with an example approach for quantification of hypertranscription is described in Example 4.
  • tumor tissue and normal tissue count differences for ENCODE-annotated cCREs is performed, wherein the ENCODE-annotated cCRES can be, for example, promoter, proximal or distal enhancer, or insulator sites,.
  • replication-coupled histone clusters are used as proxies for cell proliferation to confirm hypertranscription within the samples.
  • the presently disclosed methods allow application of data mining tools to infer gene regulatory networks.
  • a peak-caller for example, SEACR (Meers, M.P., Tenenbaum, D. & Henikoff, S. Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics & Chromatin 12, 42 (2019); doi: 10.1186/sl3072-019-0287-4), is applied to identify hypertranscribed loci throughout the genome.
  • a method of quantifying increases or decreases in RNAPII at one or more loci comprising performing a method as described herein, wherein the first affinity reagent specifically binds to a subunit of the RNAPII complex or a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII- Ser2/5, or RNAPII-Ser5/7. See, e g., Turowski TW, Boguta M. Specific Features of RNA Polymerases I and III: Structure and Assembly. Front Mol Biosci.
  • the method can further comprise comparing the results to a control reference.
  • the method may comprise direct measurements of transcription initiation by mapping and quantitating paused RNAPII to thereby characterize hypertranscription at active regulatory elements, such as promoters, enhancers, gene bodies, etc.
  • Methods can comprise quantifying increases or decreases in RNAPII relative to a control reference, for example a known value or range of values indicative of basal levels of RNAPII or amounts or presence in a tissue or a cell or populations thereof, for example a non-diseased (e.g., non-cancerous) state tissue or cell.
  • hypertranscription of a cis-regulatory element is measured as the excess of RNAPII-Ser5p in the indicated tumor over normal.
  • a method of detecting presence of a protein of interest on chromatin comprising performing a method as described herein, wherein the first affinity reagent that specifically binds to the targeted chromatin protein is specific for the protein of interest to thereby detect the presence of the protein of interest on chromatin.
  • the disclosure encompasses methods of detecting an amount of a protein of interest on chromatin, comprising performing a method as described herein, wherein the first affinity reagent that specifically binds to the targeted chromatin protein is specific for the protein of interest to thereby detect the amount of the protein of interest on chromatin.
  • the disclosure provides a method of detecting an epigenetic modification on a protein, comprising performing a method as described herein, to determine the presence of the epigenetic modification on the protein.
  • a method of detecting an epigenetic modification on a protein comprising performing a method as described herein, to determine the presence of the epigenetic modification on the protein.
  • the disclosure also encompasses methods of preparing a library of excised chromatin DNA that is amenable to sequencing on any desired platform.
  • the method comprises the steps described herein.
  • compositions that can be used in the methods described herein are also provided.
  • a composition comprises a deparaffinized and permeabilized FFPE sample containing an RNAPII specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions.
  • a composition comprises a deparaffinized and permeabilized FFPE sample containing a chromatin protein specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions.
  • the disclosure provides a kit of reagents, and optionally instructions, to facilitate performance of the methods described herein.
  • the kit comprises two or more reagents (e.g., 3, 4, 5, or more) selected from a RNAPII-specific affinity reagent, one or more chromatin protein-specific affinity reagent, a SDS solution, a Triton® X-100 (octyl phenol ethoxylate) solution, a transposase solution, a tagmentation buffer, a cross-linking reversal solution, and amine-functionalized magnetic beads.
  • reagents are described in more detail above and all embodiments thereof are encompassed by this aspect and are not repeated here in detail.
  • the kit may also comprise a low ionic solution to provide ionic conditions for transposase activity.
  • the kit can optionally include written indicia (for example labels and/or instructions) directing the performance of the method as described herein.
  • written indicia for example labels and/or instructions
  • Such labeling and/or instructions can include, for example, information concerning the amount, and method of administration, detection and quantification for the assays detailed herein.
  • ChlP-seq for chromatin profiling
  • ATAC-seq (11)
  • enzymetethering methods such as CUT&RUN (12) and CUT&Tag (13).
  • Modifications to the standard ATAC-seq protocol were required to make it suitable for FFPEs, including nuclei isolation following enzymatic tissue disruption and in vitro transcription with T7 RNA polymerase (14, 15).
  • the same group also similarly modified CUT&Tag and included an epitope retrieval step using ionic detergents and elevated temperatures, which they termed FFPE tissue with Antibody-guided Chromatin Tagmentation with sequencing (FACT-seq) (16).
  • FACT-seq is a 5-day protocol even before sequencing, and the many extra steps required relative to CUT&Tag have raised concerns about experimental variability (4).
  • Triton® -X100 was included in all buffers from antibody addition through tagmentation, which maintains cells permeable without disrupting nuclei and improves bead behavior.
  • concentration of SDS was also increased and thermolabile Proteinase K included in the fragment release buffer. After digestion at 37°C and inactivation at 58°C, the SDS is quenched with excess Triton®-X100 and the material is subjected to PCR, resulting in high yields with 30,000-60,000 cells (FIGS. 10A-10B).
  • this modified CUT&Tag-direct protocol for native whole cells resulted in representative profiles that match those of native or fixed nuclei using either the original organic extraction method or CUT&Tag-direct (FIG. 1A).
  • FiP MACS2 peakcalling and Fraction of Reads in Peaks
  • slightly more peaks called and similar FRiP values for up to at least 100,000 native whole cells were obtained using the modified protocol (FIGS. 1B-1C), obviating the need to purify nuclei for CUT&Tag-direct and AutoCUT&Tag (20).
  • Formaldehyde cross-links are reversed by incubation at elevated temperatures.
  • a relationship between cross-link reversal and incubation temperature has been determined to follow the Arrhenius equation (21).
  • Typical ChlP-seq, CUT&RUN and CUT&Tag protocols recommend cross-link reversal at 65°C overnight in the presence of proteinase K and SDS to simultaneously reverse cross-links and deproteinize.
  • the much more extreme formaldehyde treatments that are used in preparing FFPEs have required incubation temperatures as high as 90°C for isolation of PCR-amplifiable DNA for whole-genome sequencing (19, 22, 23).
  • Rhodococcus contamination an ideal calibration standard, because the two genomes are already present in the initial FFPE samples. This is unlike spike-ins used routinely for calibration of epigenomic and transcriptomic profiling, which require a mixing step that inevitably introduces stochastic errors.
  • spike-ins used routinely for calibration of epigenomic and transcriptomic profiling, which require a mixing step that inevitably introduces stochastic errors.
  • the near-perfect anti-correlation seen for these two genomes in different samples was interpreted as reflecting a very uniform distribution of contamination for slides prepared at different times.
  • Rhodococcus DNA fragments from the same tumor and naive samples almost perfectly superimposed.
  • the fragment length distribution for tumor samples is similar to that of the non-chromatinized Rhodococcus genome for >100-bp fragments when the 10-bp periodicity that is characteristic of Tn5 tagmentation is smoothed (FIG. 3B).
  • the genome in tumor cells appears to be more accessible than that in naive cells.
  • this shift to a longer fragment distribution for tumors is also seen for mitochondrial DNA from the same samples when compared to either naive brain or CUT&Tag mitochondrial DNA profiles from native 3T3 fibroblasts (FIG. 3C).
  • H3K27ac CUTAC profiles show much cleaner profiles than those obtained using FACT-seq, with higher sensitivity than the data obtained for CUT&Tag controls of frozen mouse kidney (FIGS. 4A-4D).
  • clean profiles were also seen for RNAPII- Ser2,5p FFPE-CUTAC, where RNAPII-Ser2 phosphate marks elongating and RNAPII-Ser5 phosphate marks paused RNAPII.
  • RNAPII FFPE-CUTAC profiles distinguish brain tumors [0138] Nearly all strong peaks seen for H3K27ac and RNAPII-Ser2,5p FFPE-CUTAC corresponded to putative regulatory elements from the cCRE database, with concordance between FFPE-CUTAC, FACT-seq and ChlP-seq (FIGS. 4A-4D). To identify tumor-specific candidate regulatory elements pairwise comparisons were performed between three different mouse brain tumors (YAP1-, PDGFB- and RELA-driven tumors) and normal mouse brains.
  • RNAPII-Ser5p RNAPII-Ser2,5p or H3K27ac
  • RNAPII-Ser5p + RNAPII-Ser2,5p antibody combination
  • FFPE-CUTAC can distinguish tumors from one another and from normal brains based on differences in cCRE occupancy of active RNAPII and H3K27ac marks.
  • FIGS. 6B-6C which are also from the Pdgfb-driven tumor and naive comparison, display clear differences between the tumors, with the RelA-driven tumor showing a high signal over the cCRE and the Yap 1 -driven tumor showing low signal. Even more striking differences between tumors are seen for the next two most significant differences (FIGS. 6D-6E), where the RelA-driven tumor shows a strong signal but there is no perceptible signal in the region for naive, Pdgfb-driven and Yapl-driven samples. Conspicuous tumor-specific differences are also seen for four of the five cCREs with the highest signals with FDR ⁇ 0.05 (FIGS. 6F-6J).
  • FFPE-CUTAC distinguishes tumor from normal tissue within the same FFPE
  • On-slide FFPE-CUTAC (FIG. 2A) provided us with the opportunity to compare tumor with normal tissue on the same slide.
  • ZFTA-RELA gene fusion- driven ependymomas (FIG. 8A) were used which are relatively large and cytologically distinct, whereas PDGFB-driven gliomas (FIG. 8B) are more diffuse.
  • On-slide FFPE- CUTAC were performed through tagmentation and manually harvested 6 sections from a single RELA slide and 7 sections from a single PDGFB slide separately into PCR tubes.
  • the top down-regulated gene in both replicate slides is a microRNA methylation marker locus for Helicobacter pylori infection that correlates with gastric cancer driver gene methylation 53 .
  • the entire locus is embedded in a cluster of 27 cCREs, and all replicates show a broad RNAPII signal in normal tissue but not RELA-driven tumor encompassing the entire cluster (FIG. 8D).
  • the top 10 down-regulated cCREs are either Mirl24a-hgl or Mirl24a-hg2 and these together with the next down-regulated cCRE, which is over the Mir670 microRNA locus, account for 15 of the top 25 down- regulated cCREs.
  • RNA-seq list ranked by false discovery rate, as Mirl24a-lhg ranks 9,913, Mirl24a-2hg ranks 6,045 and Mir670 ranks 21,262 of 23,551 annotated mouse genes.
  • FFPE-CUTAC distinguishes tumors from normal liver
  • FFPE-CUTAC was performed using FFPE sections prepared from intrahepatic cholangiocarcinoma tumors and normal liver. FFPE sections were used that had been fixed in formalin for 7 days and after deparaffinization were incubated at 90°C in cross-link reversal buffer for 8 hours and incubated with a 50:50 mixture of RNAPII-Ser5p and RNAPII-Ser2,5p antibodies, each at 1 :50 concentration. Highly consistent results were obtained for samples ranging from 10% to 50% of a section (-30,000-150,000 cells), with clean peaks over housekeeping genes for both liver tumor and normal liver (FIGS. 7A-7D).
  • FFPE-CUTAC provides high-quality for FFPEs from diverse tissue types.
  • RNA-seq The murine brain tumor lines that were used in the study have served as models for the study of de novo ependymoma tumorigenesis (38-40), with high-quality RNA-seq data available. To do an unbiased comparison between FFPE-CUTAC regulatory elements and processed transcripts mapped by RNA-seq, it was first determined whether there is sufficient overlap between cCREs and annotated 5’-to-3’ genes to fairly compare these very different modalities.
  • the 343,731 cCREs average 272 bp in length, accounting for 3.4% of the MmlO build of the mouse genome, whereas the 23,551 genes in RefGene average 49,602 bp in length, with an overlap of 54,062,401 bp or 2.0% of MmlO.
  • the 5’-to-3’ span of mouse genes on the RefGene list should capture all of the RNA-seq true positives and almost 60% (2.0/3.4 x 100%) of the cCREs. With most cCREs overlapping annotated mouse genes, one can directly compare FFPE-CUTAC fragment counts to RNA-seq fragment counts by asking how well they correlate with one another over genes.
  • FFPE-CUTAC provides high specificity, where significant differences between cCREs are found for up to only -0.5% of the >343,731 cCREs, almost exclusively at the upregulated corner of the volcano plots (high positive log2 fold-change, high logio FDR) (FIGS. 5A-5D).
  • -1/3 to 1/2 of 23,551 genes show significant differences between these tumorous and naive brains using RNA-seq with massive, mostly symmetrical “volcanic eruptions” (FIG. 11).
  • FFPE-CUTAC shows high promoter peaks for RelA-driven tumors and naive brain not seen in Pdgfb- and Yap 1 -driven tumors, whereas RNA-seq shows nearly the opposite, which might be an example of regulatory elements becoming accessible because of repressor binding.
  • RNA-seq shows nearly the opposite, which might be an example of regulatory elements becoming accessible because of repressor binding.
  • RNA-seq has been the go-to method for profiling the transcriptome, it only captures processed transcripts and as a result, routinely reports on a few thousands of abundant transcripts from a tissue.
  • the >300,000 genomic sites annotated as candidate cv.s-regulatory elements in the mouse genome can potentially provide direct information on transcriptional regulatory networks.
  • nucleosome-depleted regions that are mapped using accessibility methods such as ATAC-seq and CUTAC are much better suited for FFPEs, as the protein machineries that occupy these sites are not especially lysine-rich.
  • the YSPTSPS heptamer present in 52 tandem copies on the C-terminal domain of the largest subunit of RNAPII presents abundant lysine-free epitopes for CUT&Tag, and the use of low-salt tagmentation after stringent washes allows for tight binding of the Tn5 transposome within the confines of the NDR.
  • H3K27ac FFPE-CUTAC detected cCREs even more sensitively than standard H3K27ac CUT&RUN on frozen tissue, which might indicate that better reversal of cross-links at NDRs than at nucleosomes improves tagmentation within NDRs while nucleosomes remain relatively intractable.
  • Paraffin-resident DNA has the unique advantage over spike-in strategies of being present in the sample before it is processed and as a result near-perfect anti-correlations are seen with cellular DNA as they compete with one another during PCR.
  • resident Rhodococcus DNA was utilized as a size standard, allowing the conclusion that the larger size distribution of tumor relative to naive fragments has a biological basis, as the size differential was seen for both mouse nuclear and mitochondrial DNA but not for Rhodococcus DNA from the same samples.
  • paused chromatin profiling was shown to be conveniently and inexpensively performed on FFPEs in single PCR tubes. Only heat in a suitable buffer was utilized to reverse the cross-links while making the tissue sufficiently permeable, followed by needle extraction and a modified version of the CUT&Tag-direct protocol, which is routinely performed in many laboratories (18, 42). Data quality using low-salt tagmentation for antibody -tethered paused RNAPII chromatin accessibility mapping was found sufficient to distinguish cancer from normal tissues and resolve closely similar brain tumors. Using elevated levels of paused RNAPII as a discriminator, our study identified many known cancer-associated genes to be upregulated in tumors when compared to naive brain, validating our approach.
  • mice were euthanized and their brains removed and fixed at least 48 hours in neutral buffered formalin. Brains were sliced into five pieces and processed overnight in a tissue processor, mounted in a paraffin block and 10 micron sections were placed on slides. Slides were stored for varying times between 1 month to ⁇ 2 years before being deparaffinized and processed for FFPE-CUTAC. Deparaffinization was performed in Coplin jars using 2-3 changes of histology grade xylene over a 20 minute period, followed by 3-5 minute rinses in a 50:50 mixture of xylene: 100% ethanol, 100% ethanol (twice), 95% ethanol, 70% ethanol and 50% ethanol, then rinsed in deionized water. Slides were stored in distilled deionized water containing 0.02% sodium azide for up to 2 weeks before use.
  • Concanavalin A (ConA) coated magnetic beads (Bangs Laboratories, ca. no. BP531) were activated just before use with Ca ++ and Mn ++ as described (18). Frozen whole-cell aliquots were thawed at room temperature, split into PCR tubes and 5 pL ConA beads were added with gentle vortexing. All subsequent steps through to library preparation and purification followed the standard CUT&Tag-direct protocol (18), except that 1) all buffers from antibody incubation through tagmentation included 0.05% Triton®-X100; 2) the fragment release step was performed in 5 pl 1% SDS supplemented with 1 : 10 thermolabile proteinase K (New England Biolabs cat. no.
  • Tissue sections on deparaffinized slides were diced using a razor and scraped into a 1.7 mL low-bind tube containing 400 pl 800 mM Tris-HCl pH8.0, 0.05% Triton®-X100. Incubations were performed at 80-90°C for 8-16 hours or as otherwise indicated either in a heating block or divided into 0.5 mL PCR tubes after needle extraction. Needle extraction was performed either before or after Concanavalin A-bead addition using a 1 ml syringe fitted with a 1” 20 gauge needle with 20 up-and-down cycles, and in some cases was followed by 10 cycles with a 3/8” 26 gauge needle.
  • Concanavalin A ConA
  • Strong magnet stand e.g., Miltenyi Macsimag separator, cat. no. 130-092-168
  • Vortex mixer e.g., VWR Vortex Genie
  • Mini-centrifuge e.g., VWR Model V
  • Tube Rotator or Nutator e.g., VWR Model V
  • thermocycler e.g., BioRad/MJ PTC-200
  • H2O Distilled, deionized or RNAse-free H2O (dH2O e.g., Promega, cat. no. Pl 197)
  • ETA Ethylenediaminetetraacetic acid
  • BSA Bovine Serum Albumen
  • Secondary antibody e.g., guinea pig a-rabbit antibody (Antibodies online cat. no. ABIN101961) or rabbit a-mouse antibody (Abeam cat. no. ab46540)
  • PCR primers 10 pM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. et al. Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt-free primers may be used. Nextera or NEBNext primers are not recommended.
  • SPRI paramagnetic beads e.g., HighPrep PCR Cleanup Magbio Genomics cat. no. AC- 60500
  • Triton® -Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 250 pl Triton®-X100 and 12.5 pl 2 M spermidine, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA- Free tablet. Store the buffer at 4°C for up to 2 days.
  • Antibody buffer Mix 5 pl 200X BSA with 1 ml Triton®-Wash buffer and chill on ice.
  • CUTAC-DMF Tagmentation buffer Mix 780 pl dH 2 O, 200 pl N,N- dimethylformamide, 10 pl 1 M TAPS pH 8.5, 5 pl Triton® -X100 and 5 pl 1 M MgC12 (10 mM TAPS, 5 mM MgC ⁇ , 20% DMF, 0.05% Triton®-X100). Store the buffer at 4 °C for up to 1 week.
  • TAPS wash buffer Mix 1 mL dH 2 O, 10 pl 1 M TAPS pH 8.5, 0.4 pl 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA). Store at room temperature.
  • Option 1 1. Deparaffinize FFPE section affixed to slide using xylene (1 hr).
  • Option 2 Deparaffinize FFPE section affixed to slide with mineral oil.
  • Step 18 Repeat Step 18 until the interface is clear or nearly so. Using a wide-bore 200 pl pipette tip transfer 100 pl to PCR tubes.
  • ConA bead slurry Resuspend and withdraw enough of the ConA bead slurry, ensuring that there will be ⁇ 5 pl for each final sample. For example, 160 pl ConA bead slurry were added to 1.5 mL of Binding buffer for 32 samples. Place the pipette tip below the meniscus to avoid coating the beads with oil and discharge the beads while mixing by pipetting.
  • the protocol for FFPEs is similar to CUT&Tag-direct Version 3 and can be performed in parallel with native or lightly cross-linked nuclei or whole cells. Although whole cells are not appropriate with that version, including 0.05% Triton®-X100 from antibody binding to tagmentation stabilizes the bead pellet and permeabilizes cells such that by the time of tagmentation the remaining cellular material is no longer inhibitory for PCR. Now 0.05% Triton®-X100 is added by default for all CUT&Tag and CUTAC protocols, including for single cells. It was found that best results are obtained adding 1 : 10 thermo- labile proteinase K to the fragment-release solution and incubating as in this protocol pre- PCR.
  • N,N-dimethylformamide is a dehydrating compound resulting in improved tethered Tn5 accessibility and library yield.
  • Conditions used for FFPEs are the most stringent tested in Henikoff S, Henikoff JG, Kaya-Okur HS, Ahmad K. Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. Elife. 2020 Nov 16;9:e63274. doi: 10.7554/eLife.63274 - Figure 3 - figure supplement 2.
  • Cycle 2 72°C for 5 min (gap filling)
  • Cycle 4 98°C for 10 sec
  • Cycle 5 63 °C for 30 sec
  • CUT&Tag uses short 2-step 10 sec cycles to favor amplification of nucleosomal and smaller fragments.
  • DNA in FFPEs are small and PCR amplicon sizes ⁇ 120 bp are recommended (Do and Dobrovic, Clin. Chem. 61 ( 1 ):64-71 (2015)), which obviates the need to minimize the contribution of large DNA fragments.
  • Insertion of a 1 min 72°C extension and lengthening of the 63 °C annealing time from 10 sec to 30 sec results in better read-through of damaged DNA by Taq polymerase, resulting in a higher fraction of mappable reads than using the 2-step cycle favored for CUT&Tag and CUTAC.
  • Zhao L Xing P, Polavarapu VK, Zhao M, Valero-Martinez B, Dang Y, et al.
  • FACT- seq profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers. Nucleic Acids Res. 2021;49(21):el25.
  • CCAAT enhancer binding protein gamma (CZEBP- gamma): An understudied transcription factor. Advances in biological regulation.
  • Chilling device e.g., metal heat blocks on ice or cold packs in an ice cooler
  • Pipettors e.g., Rainin Classic Pipette 1 mL, 200 pL, 20 pL, and 10 pL
  • Disposable tips e.g., Rainin 1 mL, 200 pL, 20 pL
  • Vortex mixer e.g., VWR Vortex Genie
  • thermocycler e.g., BioRad/MJ PTC-200
  • RNAse-free H2O • Distilled, deionized or RNAse-free H2O (dELO e.g., Promega, cat. no. Pl 197)
  • Secondary antibody e.g., guinea pig a-rabbit antibody (Antibodies online cat. no. ABIN101961) or rabbit a-mouse antibody (Abeam cat. no. ab46540)
  • Protein A/G-Tn5 (pAG-Tn5) fusion protein loaded with double-stranded adapters with 19mer Tn5 mosaic ends (Epicypher cat. no. 15-1117)
  • NEBNext 2X PCR Master mix ME541L
  • PCR primers 10 pM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. et al. Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt-free primers may be used. We do not recommend Nextera or NEBNext primers.
  • SPRI paramagnetic beads e.g., HighPrep PCR Cleanup Magbio Genomics cat. no. AC- 60500
  • Cross-link reversal buffer Mix 800 pL 1 M Tris-HCl pH8.0, 200 pL dH2O.
  • Triton® -Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 250 pl Triton® -X100 and 12.5 pl 2 M spermidine, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4°C for up to 2 days.
  • Secondary Antibody solution Mix 17 pl guinea pig anti-rabbit (Antibodies Online) with 423 pL Triton®-Wash buffer (1 :25).
  • Protein A(G)-Tn5 solution Mix 21 pl Protein A(G)-Tn5 (Epicypher cat. no. 15-1117) with 419 pL Triton®-Wash buffer (1 :20).
  • CUTAC-DMF Tagmentation buffer Mix 17.7 mL dH 2 O, 4 mL N,N- dimethylformamide, 220 pl 1 M TAPS pH 8.5, and 110 pl 1 M MgCh (10 mM TAPS, 5 mM MgCh, 20% DMF). Store the buffer at 4°C for up to 1 week.
  • TAPS wash buffer Mix 1 mL dH 2 O, 10 pl 1 M TAPS pH 8.5, 0.4 pl 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA). Store at room temperature.
  • H3K27ac (Abeam #4729) and RNA Polymerase II Serine-2,5p (Cell Signaling Technologies CST (D1G3K) mAb #13546.
  • RNA Polymerase II Serine-2,5p Cell Signaling Technologies CST (D1G3K) mAb #13546.
  • FFPEs The protocol for FFPEs is similar to CUT&Tag-direct Version 4, available at dx.doi.org/10.17504/protocols.io.x54v9mkmzg3e/v4, and can be performed in parallel with native or lightly cross-linked nuclei or whole cells.
  • Option 2 Tagmentation (1.5 hr)
  • N,N-dimethylformamide is a dehydrating compound resulting in improved tethered Tn5 accessibility and library yield.
  • Conditions used for FFPEs are the most stringent tested in Henikoff S, Henikoff JG, Kaya-Okur HS, Ahmad K. Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. Elife. 2020 Nov 16;9:e63274. doi: 10.7554/eLife.63274 - Figure 3 - figure supplement 2.
  • CUT&Tag uses short 2-step 10 sec cycles to favor amplification of nucleosomal and smaller fragments.
  • DNA in FFPEs are small and PCR amplicon sizes ⁇ 120 bp are recommended (Do and Dobrovic, Clin. Chem. 61 ( 1 ):64-71 (2015)), which obviates the need to minimize the contribution of large DNA fragments.
  • Insertion of a 1 min 72°C extension and lengthening of the 63 °C annealing time from 10 sec to 30 sec results in better read-through of damaged DNA by Taq polymerase, resulting in a higher fraction of mappable reads than using the 2-step cycle favored for CUT&Tag and CUTAC.
  • Materials ⁇ Chilling device e.g., metal heat blocks on ice or cold packs in an ice cooler
  • Pipettors e.g., Rainin Classic Pipette 1 mL, 200 pL, 20 pL, and 10 pL
  • Disposable tips e.g., Rainin 1 mL, 200 pL, 20 pL
  • Strong magnet stand e.g., Miltenyi Macsimag separator, cat. no. 130-092-168
  • Vortex mixer e.g., VWR Vortex Genie
  • thermocycler e.g., BioRad/MJ PTC-200
  • Bio-Mag Plus amine magnetic beads (48 mg/ml, Polysciences cat. no. 86001-10). Dilute 1 : 10 with 10 mM Tris pH8/l mM EDTA for use.
  • RNAse-free H2O Distilled, deionized or RNAse-free H2O (dELO e.g., Promega, cat. no. Pl 197)
  • H3375 Hydroxy ethyl piperazineethanesulfonic acid pH 7.5
  • Triton® X-100 (Sigma- Aldrich, cat. no. XI 00)
  • ⁇ PCR primers 10 pM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. et al. Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt- free primers may be used. We do not recommend Nextera or NEBNext primers.
  • Cross-link reversal buffer 8 ml 1 M Tris-HCl pH8.0, 2 ml dH2O and 4 pl 0.5 mM EDTA.
  • Rinse buffer (Option 1) Mix 1 mL 1 M HEPES pH 7.5 and 1.5 mL 5 M NaCl, and bring the final volume to 50 mL with dH2O.
  • Triton® -Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 250 pl 10% Triton® -X100, 12.5 pl 2 M spermidine and 20 pl 0.5 M EDTA, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4°C for up to 2 days.
  • Protein A(G)-Tn5 solution Mix 21 pl Protein A(G)-Tn5 (Epicypher cat. no. 15-1117) with 419 pL Triton®-Wash buffer (1 :20).
  • CUTAC-DMF Tagmentation buffer Mix 17.7 mL dH 2 O, 4 mL N,N- dimethylformamide, 220 pl 1 M TAPS pH 8.5, and 110 pl 1 M MgCh (10 mM TAPS, 5 mM MgCh, 20% DMF). Store the buffer at 4°C for up to 1 week.
  • TAPS wash buffer Mix 1 mL dH 2 O, 10 pl 1 M TAPS pH 8.5, 0.4 pl 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA). Store at room temperature.
  • Option 1 On-slide FFPE-CUTAC deparaffinization in hot cross-link reversal buffer.
  • Option 1 On-slide FFPE-CUTAC Incubation with primary antibody [0366] 5 For each slide, remove from slide holder, wick off excess liquid from the glass surface with a Kimwipe (without touching the tissue) and place tissue-side up on a dark surface for visibility. Carefully pipette ⁇ 50 pl primary antibody solution over the tissue. [0367] 6. Cover the clear portion of the slide with a rectangle of plastic film (or a square for small tissue sections) using surface tension to spread the liquid, while excluding large bubbles and wrinkles. Place wrapped slides separated in a dry slide holder (FIG. 20) or in the rack of a staining dish, which can be used as a "moist chamber" (FIG. 25).
  • Option 1 Incubation with secondary antibody ( 1.5 hr)
  • FFPE slide or curl Scrape all or part of a 10 pm FFPE slide (FIGS. 20, 22 and 25) or a "curl" (FIG. 26) into a 1.7 ml tube (e.g., MCT-175-C), add 200 pL mineral oil. Vortex, spin, and place in a 90°C water bath for 5 min. While still warm vortex to fully suspend the paraffin and spin on full.
  • the Option 2 protocol is for 16 samples but can be scaled up or down as needed. Sequencing-ready purified DNA libraries can be obtained in one long day ( ⁇ 10 hours), but any of the 1 hr antibody or pAG-Tn5 incubations can be extended to a few hours at room temperature or at 4-8°C overnight.
  • Curls are thin sections that are released from the microtome without being affixed to slides and curl up to form a tight rod.
  • Hypertranscription is global upregulation of transcription, which is common in rapidly proliferating cells. In cancer, hypertranscription confers a worse prognosis, independent of somatic mutation burden, tumor ploidy, tumor stage, patient gender, age, or tumor subtype (Zatzman et al. Sci Adv 8(47):eabn0238 (2022). Hypertranscription has thus far been assayed indirectly using RNA-seq data calibrated in variety of ways, but none have been suitable for clinical application.
  • FFPE-CUTAC can be used to directly map hypertranscription at regulatory elements throughout the mouse genome, revealing that the degree of hypertranscription varies between genetically identical tumors and for some is not observed at all.
  • FFPE-CUTAC analyzed for hypertranscription identified dozens of strongly hypertranscribed loci in common among the tumors. Strikingly, in two of the seven individual tumors broad increases of RNAPII within chromosome 17ql2-21, which includes WIQ ERBB2 (HER2) locus, were observed.
  • HER2 amplifications were punctuated with broad hypertranscribed regions, suggestive of linkage disequilibrium during tumor evolution (17, 18).
  • Our data suggest that selective sweeps of direct regulators of RNAPII, including the CDK12 kinase, contribute to the poor prognosis associated with hypertranscription.
  • FFPE-CUTAC to categorize tumors with sparse material, precisely localize patterns of regulatory element hypertranscription, and map megabase-sized regions of amplification interspersed with smaller regions of likely clonal selection, makes it an attractive platform for general personalized medicine applications.
  • Upregulation bias based on RNAPII log2(fold- change) plotted on the -axis as a function of loglO(average signal) on the x-axis (MA plot) is observed in pooled data from several experiments in which tumor-rich sections were separated from normal sections (FIGS. 35A-35E).
  • the upregulation bias is much greater for RELA than for PDGFB, and to further understand these differences and to eliminate sample- to-sample variability, on-slide dissection data from single FFPE slides representing normal mouse brain, RELA and YAP1 tumors and PDGFB tumors from two genetically identical mice were examined.
  • upregulation based on foldchange showed little relationship to the percentage of tumor in the sample as determined by counting cells stained for tumor transgene expression.
  • YAP1 tumor sections averaged 16% tumor cells and showed similar upregulation bias to the PDGFB- 1 sections with 80% tumor cells and stronger upregulation bias than the PDGFB-2 sections with 64% tumor cells (FIGS. 35F- 35H, right panels) and all three showed weaker upregulation versus the RELA tumor sections with 40% tumor cells (FIGS. 35F-35H, left panels).
  • the fold-change ratio of tumormormal does not distinguish between a weak signal increasing to moderate strength and a moderate signal increasing to high strength.
  • RNAPII FFPE-CUTAC assay is well-suited to detect minor absolute differences in regulatory element RNAPII occupancy (FIG. 28A), unlike RNA readouts that require calibration to the DNA template. For each tumor and normal sample, the number of mapped fragments spanning each base-pair in a cCRE scaled to the mouse genome were counted and the number of counts over that cCRE averaged.
  • these small single-exon genes produce RNAPII-dependent U7-processed single-exon mRNAs during S-phase to encode for the histones that package the entire genome in nucleosomes, and so the abundance of RNAPII at these histone gene loci provides a proxy for steady-state DNA synthesis genome-wide.
  • 54 are within the major histone gene cluster on Chromosome 13, and when Tumor and Normal dissection data from multiple experiments are displayed, differences are seen between tumor samples consistent with the observation of RNAPII hypertranscription differing between samples (FIG. 28F).
  • RNAPII-Ser5p FFPE-CUTAC was performed, and each pair rank- ordered by Tumor minus Normal differences to test for RNAPII hypertranscription based on the 984,834 ENCODE-annotated human cCREs.
  • cCREs in repeat-masked regions of the hgl9 build were removed, the data pooled from all four independent experiments and equalized the number of fragments between tumor and normal samples.
  • FFPE-CUTAC and other tagmentation methods non-specifically recover a small fraction of mitochondrial DNA (mtDNA, Chromosome M) due to the enhanced accessibility of nucleosome-free mtDNA.
  • RNAPII- Ser5p FFPE-CUTAC detected a much lower level of mtDNA in most tumor samples than in their matched normal samples for both mouse and human (FIGS. 30A-30B), suggesting that these tumors contain fewer mitochondrial genomes.
  • publicly available ATAC-seq data from both the TCGA and ENCODE projects were mined.
  • TCGA tumor data the percentage of mtDNA ranges from -4% for glioblastoma, a brain cancer, -25% for adrenal carcinoma, whereas for ENCODE data, which are from healthy individuals, percentages range from -1% for kidney to -21% for brain (FIGS. 30C-30D).
  • This 6- fold higher level of mitochondrial ATAC-seq signal in normal brain in the ENCODE data over that of glioblastoma in the TCGA data is consistent with decreased mitochondrial DNA abundance in most human and mouse tumors in the FFPE-CUTAC data.
  • These reductions in mtDNA by both CUTAC and ATAC-seq are consistent with reductions in mtDNA reported based on whole-genome sequencing (24), suggestive of relaxed selection for maintenance of mtDNA in cancer.
  • SEACR Sese Enrichment Analysis for CUT&RUN
  • SEACR optionally uses a background control dataset, typically for a non-specific IgG antibody.
  • the background control was replaced with the normal sample in each pair, merged fragment data, duplicates removed and read numbers equalized for the seven human Tumor/Normal pairs.
  • SEACR reported a median of 4483 peaks, and when Tumor and Normal were exchanged, a median of only 15 peaks was reported, which suggests that hypertranscription is more common than hypotranscription. Therefore, SEACR Tumor/Normal peaks can be used as an unbiased method for discovering the most hypertranscribed loci in the human cancer samples.
  • SEACR Tumor/Normal peak calls corresponded to the 100 top-ranked cCREs in the overall list representing all seven tumors.
  • all 100 cCREs at least partially overlapped one or more SEACR Tumor/Normal peak call, and in addition, the large majority of the 100 top-ranked cCREs intersected with overlapping SEACR peak calls from multiple Tumor/Normal pairs (Table 2).
  • Each of the #l-ranked cCREs in the Br, Co, Li, Lu and Re tumor samples respectively intersected MSL1, RFFL, PABPC1, CLTC and SERINC5 genes and also overlapped SEACR peak calls in 4-5 of the 7 tumors (FIGS. 31A-31E).
  • the #l-ranked cCRE in the St sample intersected an intergenic enhancer in the HSP90AA1 gene and overlapped SEACR peak calls in both Br and St (FIG. 31F).
  • No SEACR peaks were observed for the kidney sample, as expected given the lack of detectable cCRE or histone locus hypertranscription.
  • the large majority of strongly RNAPII-hypertranscribed regulatory elements are hypertranscribed in multiple human cancers.
  • RNAPII-Ser5p FFPE-CUTAC revealed that the hypertranscription differences between liver tumors from unrelated individuals conspicuously differed. For example, all four cCREs that ranked #1 and #2 in either liver tumor showed strong hypertranscription in the first liver tumor but only weak hypertranscription in the second (FIGS. 32A-32D), and similar results were observed for the replication-coupled histone genes (FIG. 32E). Hypertranscription for the top-ranked >10,000 cCREs was observed for both liver tumor samples, again much stronger for the first tumor than for the second (FIG. 32F).
  • stomach tumor cluster comprised four samples from four different experiments with a median of -470,000 mapped fragments (FIG. 33B).
  • the Co and Br tumor samples clustered very close to one another, suggesting that this pair of individual tumors share oncogenic loci to a much greater extent than would be expected for such different tissue types.
  • amplification of a region will appear as a proportional increase in the level of RNAPII over the amplified region, so that one can interpret regional hypertranscription in both the Br and Co tumor samples as revealing independent amplification events.
  • each of the six summits in the Chrl7ql2-21 region in the Br tumor sample were superimposed over the raw data tracks on expanded scales for clarity, centered over the highest promoter peak in the region (FIG. 34F).
  • the -100 kb broad summit is almost precisely centered over the -1 kb wide ERBB2 promoter peak.
  • each summits are less broad, each is similarly centered over a promoter peak.
  • our results are inconsistent with independent upregulation of promoters over the HER2 amplified regions. Rather, it appears that a HER2 amplification event was followed by clonal selection for broad regions around ERBB2 and other loci within each amplicon.
  • MED1 encodes a subunit of the 26-subunit Mediator complex, which regulates RNAPII pause release
  • CDK12 is the catalytic subunit of the CDK12/Cyclin K kinase heterodimer complex, which phosphorylates RNAPII on Serine-2 for productive transcriptional elongation.
  • Cyclin K is the regulatory subunit of the CDK12 kinase
  • the CCNK gene that encodes Cyclin K would be strongly upregulated in the Br tumor but not necessarily in the Co tumor. Indeed, we see a 5.4-fold increase in RNAPII-S5p over the CCNK promoter in the Br tumor relative to adjacent normal tissue, whereas in the Co tumor there is a 2.1 -fold increase (FIG. 34C), consistent with RNAPII hypertranscription directly driven in part by CDK1 amplification.
  • FFPE-CUTAC takes advantage of the hyperaccessibility and abundance of the targeted epitope and the impermeability of histone cross-linked chromatin to achieve exceptional signal- to-noise.
  • RNAPII FFPE-CUTAC maps the transcriptional machinery itself directly on the DNA regulatory elements, direct measurements of transcription initiation were obtained, as opposed to inferences based on estimating steady-state mRNA abundances.
  • our mapping and quantitation of paused RNAPII a critical checkpoint between transcriptional initiation and elongation, represents a powerful general approach to characterize hypertranscription at active regulatory elements genome-wide.
  • SEACR identified all of the 100 top-ranked of nearly 1 million human cCREs in at least one tumor (Table 2), reporting a median of 3.7 overlapping cCREs in six of the seven different human tumors in our study. Reductions in mitochondrial DNA that varied between tumors were also observed, suggestive of relaxed selection for mtDNA-encoded products during cancer progression.
  • HER2 amplifications are known to be subject to clonal selection, resulting in tumor heterogeneity (31), consistent with our observation of broad summits centered directly over promoters of candidate cancer driver genes within the amplified regions.
  • FFPE- CUTAC thus potentially provides a general diagnostic strategy for detection and analysis of amplifications and clonal selection during cancer progression and therapeutic treatment.
  • CDK12 a cyclin-dependent kinase that phosphorylates RNAPII on Serine-2 for pause release and transcriptional elongation and which is co-amplified with HER2 in -90% of HER2 + breast cancers (35).
  • Cyclin K the regulatory subunit of the CDK12/Cyclin K kinase complex is strongly upregulated in the same tumor, which suggests that amplification of CDK12 directly contributes to RNAPII hypertranscription and is in part responsible for poor prognosis in HER2/CDK12-amplified breast cancer patients (28, 35, 36).
  • FFPE-CUTAC to cohorts of HER2-amplified and other cancer patient samples is envisioned to ascertain the generality of the model for hypertranscription.
  • RNAPII and H3K27ac epitopes used in FFPE-CUTAC have made possible detection of genome-wide hypertranscription using single 5 pm thick FFPE tissue sections -1 cm 2 in area and fewer than 4 million unique fragments.
  • Our identification of HER2 amplifications and probable clonal selection events that did not rely on reference to any external data emphasizes the potential power of our approach for understanding basic genetic and epigenetic mechanisms underlying tumor evolution.
  • the simple workflow of FFPE-CUTAC and its potential for scale-up and automation make it an attractive platform for retrospective studies and will require little modification for routine cancer screening and other personalized medicine applications.
  • mice were injected intracranially with DF1 cells infected with and producing RCAS vectors encoding either PDGFB (21), ZFTA-RELA (19), or YAP1- FAM1 18b (20) as has been described (37).
  • RCAS vectors encoding either PDGFB (21), ZFTA-RELA (19), or YAP1- FAM1 18b (20) as has been described (37).
  • mice Upon weaning (-P21), mice were housed with same-sex littermates, with no more than 5 per cage and given access to food/water ad libitum. When the mice became lethargic and showed poor grooming, they were euthanized and their brains removed and fixed at least 48 hours in neutral buffered formalin.
  • RNAPII-Ser5p Cell Signaling Technologies cat. no. 13523, lot 3
  • RNAPII-Ser2p Cell Signaling Technologies cat. no. 13499
  • H3K27ac Abeam cat. no. ab4729, lot no. 1033973.
  • Secondary antibody Guinea pig a-rabbit antibody (Antibodies online cat. no. ABIN101961, lot 46671).
  • the sections were immediately covered with 20-60 pL primary antibody in Triton®-Wash buffer (20mM HEPES pH 7.5,150mMNaCl, 2mM spermidine and Roche complete EDTA-free protease inhibitor) added dropwise.
  • Plastic film was laid on top to cover and slides were incubated >2 hr incubation at room temperature (or overnight at ⁇ 8°C) in a moist chamber. The plastic film was peeled back, and the slide was rinsed once or twice by pipetting 1 mL Triton®-Wash buffer on the surface, draining at an angle. This incubation/wash cycle was repeated for the guinea pig antirabbit secondary antibody (Antibodies Online cat. no.
  • Cutadapt 2.9 (40) was used with parameters "-j 8 — nextseq-trim 20 -m 20 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA - A (SEQIDNO:) AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -Z" (SEQIDNO:) to trim adapters from 50bp paired-end reads fastq files.
  • Bedtools 2.30.0 "genomecov" was used to make a normalized count track which is the fraction of counts at each base pair scaled by the size of the reference sequence so that if the counts were uniformly distributed across the reference sequence there would be one at each position.
  • SEACR 1.3 (25) was run with parameters "norm relaxed" on tumor samples with the normal sample from each tumor and normal pair as the control. For comparison, we also called peaks after reversing the roles of tumor and normal.
  • TF-IDF frequency-inverse document frequency
  • Custom scripts used in this study are available from GitHub: github . com/Henikoff/FFPE .
  • c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell. 2012; 15 l(l):68-79. 5. Lin CY, Loven J, Rahl PB, Paranal RM, Burge CB, Bradner JE, et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell. 2012; 151 (1): 56-67.
  • Chilling device e.g. metal heat blocks on ice or cold packs in an ice cooler
  • Pipettors e.g. Rainin Classic Pipette 1 mL, 200 pL, 20 pL, and 10 pL
  • Disposable tips e.g. Rainin 1 mL, 200 pL, 20 pL
  • Disposable centrifuge tubes for reagents 15 mL or 50 mL
  • thermocycler e.g. BioRad/MJ PTC-200
  • Safe Clear II Fisher cat. no. 23-044192
  • Bio-Mag Plus amine magnetic beads 48 mg/ml, Polysciences cat. no. 86001-10). Dilute 1: 10 with 10 mM Tris pH8/l mM EDTA for use.
  • PCR primers 10 pM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. et al. Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt-free primers may be used. We do not recommend Nextera or NEBNext primers.
  • SPRI paramagnetic beads e.g. HighPrep PCR Cleanup Magbio Genomics cat. no. AC- 60500
  • Rinse buffer (Option 1) Mix 1 mL 1 M HEPES pH 7.5 and 1.5 mL 5 M NaCl, and bring the final volume to 50 mL with dH2O.
  • Triton-Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 250 pl 10%
  • Triton-XlOO 12.5 pl 2 M spermidine, bring the final volume to 50 mL with dH2O, and add 1
  • Protein A(G)-Tn5 solution Mix 21 pl Protein A(G)-Tn5 (Epicypher cat. no. 15-1117) with 419 pL Triton-Wash buffer (1 :20).
  • CUTAC-DMF Tagmentation buffer Mix 17.7 mL dH2O, 4 mL N,N-dimethylformamide, 220 pl 1 M TAPS pH 8.5, and 110 pl 1 M MgC12 (10 mM TAPS, 5 mM MgC12, 20% DMF).
  • TAPS-EDTA wash buffer Mix 1 mL dH2O, 10 pl 1 M TAPS pH 8.5, 0.4 pl 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA). Store at room temperature.
  • the Option 1 protocol is for 16 samples but can be scaled up or down as needed.
  • the example experiment shown in FIGS. 22, 23 and 27 beginning with dry FFPE slides through sequencing-ready purified DNA libraries was accomplished in one long day ( ⁇ 11 hours), but all of the steps can be lengthened with proper sealing to minimize evaporation. Overnight stopping points can be during any of the room temperature incubations by placing the plastic film- wrapped slides into a moist chamber and holding at 4-8 °C.
  • Option 1 Incubation with secondary antibody ( 1.5 hr).
  • FFPE slide or curl Scrape all or part of a 5-10 pm FFPE slide (FIGS. 20, 22, 25) or a "curl" (FIG. 26) into a 1.5-2 ml tube (e.g., MCT-175-C). Add 320 pl Safe Clear II. Vortex, spin, and place in a 56°C water bath for 3 min. Cool and centrifuge on full for 2 min.
  • the Option 2 protocol is for 16 samples but can be scaled up or down as needed. Sequencing-ready purified DNA libraries can be obtained in one long day ( ⁇ 10 hours), but any of the 1 hr antibody or pAG-Tn5 incubations can be extended to a few hours at room temperature or at 4-8°C overnight.
  • Curls are thin sections that are released from the microtome without being affixed to slides and either curl up to form a tight rod (10 pm) or fold (5 pm). Best permeabilization is obtained with 5 pm curls.
  • 90°C incubations can be extended for several hours or overnight without noticeable consequences.
  • room temperature incubations with affinity reagents can be extended up to overnight by performing at 4-8°C. Differences for longer room temperature or cold incubation times have not been noticed and times less than 1 hr, have not been tested which might be OK for shortening this protocol to fit into a single day.
  • Bio-Mag Plus amine magnetic beads are -1.5 micron in diameter and have a rough hydrophilic surface that sticks weakly to deparaffinized tissue shards (FIG. 23).
  • Pierce glutathione magnetic agarose beads are 10-40 micron but are inert and don't appear to stick, although they trap the tissue as they as they migrate in a magnetic field. In a magnetic field, the combination rapidly forms a tight pellet that is not disrupted by the pipette when decanting the supernatant.
  • Option 2 Incubation with primary antibody [0533] 27. Resuspend beads in 100 pl primary antibody solution followed by vortexing.
  • FFPEs The protocol for FFPEs is similar to CUT&Tag-direct Version 4 and can be performed in parallel with native or lightly cross-linked nuclei or whole cells.
  • N,N-dimethylformamide is a dehydrating compound resulting in improved tethered Tn5 accessibility and library yield.
  • a 55°C incubation used for FFPEs is the most stringent tested in Henikoff S, Henikoff JG, Kaya-Okur HS, Ahmad K. Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. Elife. 2020 Nov 16;9:e63274. doi: 10.7554/eLife.63274 ( Figure 3 - figure supplement 2).
  • volumes here and below are calculated based on assuming that the tissue amount is equivalent to half that of a 10 micron FFPE slide or curl. Except for the sequencing primers, volumes may be scaled accordingly for different amounts of tissue.
  • Cycle 1 58°C for 5 min (gap filling)
  • Cycle 2 72°C for 5 min (gap filling)
  • Cycle 5 63°C for 30 sec
  • Cycle 6 72°C for 1 min Repeat Cycles 4-6 11 times Hold at 8 °C
  • CUT&Tag uses short 2-step 10 sec cycles to favor amplification of nucleosomal and smaller fragments.
  • DNA in FFPEs are small and PCR amplicon sizes ⁇ 120 bp are recommended (Do and Dobrovic, Clin. Chem. 61 (l):64-71 (2015)), which obviates the need to minimize the contribution of large DNA fragments.
  • Insertion of a 1 min 72 °C extension and lengthening of the 63 °C annealing time from 10 sec to 30 sec results in better read -through of damaged DNA by Taq polymerase, resulting in a higher fraction of mappable reads than using the 2-step cycle favored for CUT&Tag and CUTAC.

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Abstract

L'invention concerne des procédés in situ améliorés pour cartographier l'emplacement d'une protéine sur la chromatine et des procédés in situ se basant sur l'ADN pour mesurer la transcription dans une cellule à partir d'un échantillon fixé au formol et inclus en paraffine (FFPE). Les procédés peuvent comprendre des étapes consistant à traiter l'échantillon FFPE pour éliminer la paraffine ; perméabiliser l'échantillon ; mettre en contact l'échantillon avec un premier réactif d'affinité qui se lie spécifiquement à une protéine de chromatine ciblée ou à une protéine impliquée dans la régulation de la transcription, le premier réactif d'affinité étant couplé à un transposome comprenant au moins une transposase et un transposon, ce qui permet de cliver et de marquer l'ADN de la chromatine ; exciser le segment d'ADN marqué associé à la protéine ou à la protéine de chromatine ciblée impliquée dans la régulation de la transcription ; et déterminer la séquence nucléotidique du segment d'ADN marqué excisé, ce qui permet de mapper l'emplacement génomique de la protéine ciblée sur la chromatine ou l'activité transcriptionnelle sur la chromatine.
PCT/US2024/031983 2023-06-02 2024-05-31 Analyse épigénomique d'échantillons fixés au formol et inclus en paraffine Pending WO2024249846A2 (fr)

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