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WO2024229044A1 - Procédés de capture de conformation de chromatine sans ligature avec séquençage à haut débit - Google Patents

Procédés de capture de conformation de chromatine sans ligature avec séquençage à haut débit Download PDF

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WO2024229044A1
WO2024229044A1 PCT/US2024/027099 US2024027099W WO2024229044A1 WO 2024229044 A1 WO2024229044 A1 WO 2024229044A1 US 2024027099 W US2024027099 W US 2024027099W WO 2024229044 A1 WO2024229044 A1 WO 2024229044A1
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chromatin
dna sequences
telomere
dna
crosslinking agent
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WO2024229044A9 (fr
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Floris P. BARTHEL
Yi-An Chen
Noelle Fukushima
Ogechukwu MDEGBU
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Translational Genomics Research Institute TGen
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Translational Genomics Research Institute TGen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • Telomeres are repetitive DNA sequences at the ends of chromosomes that protect the integrity of the genome and shorten with increasing age. Telomeres can silence gene expression by spreading its heterochromatin structure to neighboring regions, i.e., by telomere position effect (TPE) or via long-range chromatin interactions over larger distances, i.e., TPE over long distances (TPE-OLD). Recent studies have nominated genes in human cells controlled by TPE- OLD in a telomere length-dependent manner, suggesting that TPE-OLD is an epigenetic mechanism of transcriptional regulation important in aging and age-related disease. However, the larger-scale structure of telomeres and their interactions with the rest of the genome remain largely unknown.
  • telomere 3D in situ hybridization A current standard for validation of telomeric chromatin interaction is telomere 3D in situ hybridization. However, its scale is limited by the number of probes applied in each experiment. SUMMARY A need exists to determine the underlying molecular mechanisms regulating telomerase reverse transcriptase (TERT) expression in cancer and other diseases. To fulfill the emerging demand for large-scale studies of telomeric chromatin interactions, the present disclosure includes methods of telomere chromatin conformation capture with high throughput sequencing for this purpose. Telomeric chromatin is densely packed with nucleosomes and shelterin.
  • Telomeres can influence gene expression by forming long-range chromatin loops (telomere position effect over long distances, or TPE-OLD). Telomerase reactivation is a fundamental event in the genesis of nearly every human cancer. Although transcriptionally silent in differentiated adult cells, its catalytic component telomerase reverse transcriptase (TERT) is expressed in over 80% of human cancers. Despite the recent discovery of reactivating TERT promoter mutations, very little is known about mechanisms leading to the reactivation of telomerase in human cancer. This disclosure provides methods for isolating associated DNA from reverse crosslinked supernatant to achieve a ligation-free library preparation.
  • the disclosed methods further capture telomere-associated complexes from crosslinked chromatin and sequence them using high-throughput sequencing. These methods enable identification of locus-to-locus interaction in a 3D genomic manner. Unlike other 3D genomic technologies, the present method is ligation-free and utilizes probes to capture chromatin with specific loci DNA. This method enables the enrichment of any desired locus and its interacting loci, offering researchers a robust tool for studying the interactions between two loci, providing a more accurate representation of chromatin interaction. The ability to enrich specific loci with this technology has the potential to offer new insights into gene regulation, chromatin structure, and genome function, making it a promising technique for researchers in a variety of fields.
  • the method of enriching a DNA locus in chromatin may include the steps of preparing chromatin by dual crosslinking using methanol-free formaldehyde and ethylene glycol bis(succinimidyl succinate) (EGS) to produce dual crosslinked chromatin, and shearing the dual crosslinked chromatin using sonication.
  • EGS ethylene glycol bis(succinimidyl succinate)
  • the Docket No.91482.266WO-PCT method may include capturing PNA probes and a set of DNA sequences from the chromatin by, (a) hybridizing peptide nucleic acid (PNA) probes with attached biotinylated beads to DNA sequences in the dual crosslinked chromatin, (b) reversing the dual crosslink in the chromatin using a protein digestion enzyme to release the DNA sequences to produce released DNA sequences, and (c) separating the released DNA sequences by applying a magnet to the biotinylated beads.
  • the method may further include the step of extracting from the mixture a fluid (supernatant) containing the released DNA sequences. The fluid does not contain the PNA probes or biotinylated beads.
  • the method may further include the steps of purifying the released DNA sequences, and generating a ligation-free library with the released DNA sequences.
  • the released DNA sequences include the DNA locus.
  • the method may further include the steps of indexing the ligation-free library, and sequencing the ligation-free library using next generation sequencing.
  • the disclosure provides a method of enriching a DNA locus in chromatin, comprising: preparing chromatin by dual crosslinking using a nucleic acid crosslinking agent and a protein crosslinking agent to produce dual crosslinked chromatin; shearing the dual crosslinked chromatin using enzymatic digestion or mechanical force; capturing peptide nucleic acid (PNA) probes and a set of DNA sequences from the chromatin by: (a) hybridizing PNA probes with attached biotinylated beads to DNA sequences in the dual crosslinked chromatin, (b) reversing the dual crosslink in the chromatin using a protein digestion enzyme to release the DNA sequences to produce released DNA sequences, and (c) separating the released DNA sequences by applying a magnet to the biotinylated beads; extracting from the mixture a fluid containing the released DNA sequences, wherein the fluid does not contain the PNA probes or biotinylated beads; purifying the released DNA sequences; generating a
  • the protein crosslinking agent is selected from the group consisting of bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), N-hydroxysuccinimide, N-hydroxysulfosuccinimide, ethylene glycol bis (succinimidyl succinate) (EGS), sulfo-ethylene glycol bis(succinimidylsuccinate) (sulfo-EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (SEGS), glutaraldehyde, Docket No.91482.266WO-PCT polyfunctional aziridine, bifunctional carbodiimide, dicyclohexyl carbodiimide
  • the nucleic crosslinking agent is selected from the group consisting of glutaraldehyde, formaldehyde, psoralen, aminomethyltrioxsalen, cisplatin, disuccinimidyl glutarate, formalin, UV light, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis diaminedichloroplatinum(II), and cyclophosphamide.
  • the protein crosslinking agent is EGS.
  • the nucleic crosslinking agent is methanol-free formaldehyde.
  • the dual crosslinked chromatin is sheared with mechanical force using sonication, nebulization, hydrodynamic shearing, syringe pumping.
  • the protein digestion enzyme is a serine protease, cysteine protease, aspartic protease, threonine protease, metalloprotease, glutamic protease, thiol protease, or a combination thereof.
  • the protein digestion enzyme is a serine protease.
  • the released DNA sequences are associated with a transposable element or interspersed repeat.
  • the transposable element or interspersed repeat is selected from the list presented in FIG. 14.
  • the transposable element or interspersed repeat is a telomere-associated DNA sequence.
  • the telomere-associated DNA sequence is a Telomere-Associated Repeat Element (TARE), Subtelomeric Repeat, Interstitial Telomeric Sequence (ITS), Short Interspersed Nuclear Element (SINE), Long Interspersed Nuclear Element (LINE), or satellite DNA.
  • TARE Telomere-Associated Repeat Element
  • ITS Interstitial Telomeric Sequence
  • SINE Short Interspersed Nuclear Element
  • LINE Long Interspersed Nuclear Element
  • satellite DNA In another aspet, the telomere- associated DNA sequence is (CCCTAA)n or (TTAGGG)n.
  • the released DNA sequences are associated with a Retrotransposable Element (RE).
  • the RE is an Alu element (ALU).
  • the ALU belongs to a subfamily selected from the group consisting of AluJ, AluS, AluY, AluSc, AluSq, AluSp, AluYb8, AluYa5, AluYh9, and AluJo.
  • the PNA probe comprises a complementary nucleotide sequence to a transposable element, interspersed repeat, or retrotransposable element.
  • the disclosure provides a method of capturing telomere-associated DNA loci from chromatin, comprising: crosslinking the chromatin by mixing chromatin with a nucleic acid crosslinking agent and a protein crosslinking agent to produce dual crosslinked chromatin; shearing the dual crosslinked chromatin using enzymatic digestion or mechanical force; capturing PNA probes and a set of DNA sequences from the chromatin by: (a) hybridizing PNA probes with biotinylated beads to DNA sequences in the dual crosslinked Docket No.91482.266WO-PCT chromatin, (b) reversing the dual crosslink in the chromatin using a protein digestion enzyme to release the DNA sequences to produce released DNA sequences, and (c) separating the released DNA sequences by applying a magnet to the biotinylated beads; and extracting from the mixture a fluid containing the released DNA sequences, wherein the fluid does not contain the PNA probes or biotinylated beads, and where
  • FIG. 1 illustrates principals and methods of ligation-free chromatin conformation capture with high throughput sequencing
  • FIG.2 illustrates details of the PNA capture probe step from FIG.1
  • FIG. 3 illustrates principals and methods of ligation-free chromatin conformation capture with high throughput sequencing
  • FIGS. 4A-4B illustrate results showing negative association between the method’s signals and chromosome ends;
  • FIGS. 1 illustrates principals and methods of ligation-free chromatin conformation capture with high throughput sequencing
  • FIG.2 illustrates details of the PNA capture probe step from FIG.1
  • FIG. 3 illustrates principals and methods of ligation-free chromatin conformation capture with high throughput sequencing
  • FIGS. 4A-4B illustrate results showing negative association between the method’s signals and chromosome ends
  • FIGS. 6A-5D illustrate results showing telomeric chromatin interacts in the loci of TERT, CCND2, ITS, and at consensus sites; Docket No.91482.266WO-PCT
  • FIGS. 6A-5D illustrate, at the end of chromosomes, the periodicity of the signal produced by the disclosed ligation-free chromatin capture and sequencing method
  • FIGS.7A-7C illustrate enrichment of the signal produced by the disclosed ligation-free chromatin capture and sequencing method in ITS regions
  • FIG.8 illustrates a representation of a telomer
  • FIGS.9A-9C illustrate example fragment yields and size quality used in the sonication quality control (QC) step of the ligation-free chromatin capture and sequencing method.
  • FIG. 10 illustrates detection of proximal and distal telomere-chromatin interaction in Chr20 by Telomere-C.
  • FIGS. 11A-11C illustrate Telomere-C signals are rich in the Telomere Associated Repeat 1, (CCCTAA)n, or (TTAGGG)n, and other repeat elements.
  • FIGS.12A and 12B illustrate validation of Telomere-ITSs interaction by 3C.
  • FIGS. 13A and 13B illustrate detection of a trans Telomere-ITS interaction by fluorescence in situ hybridization (FISH).
  • FIG. 14 illustrates a list of transposable elements or interspersed repeats that can be analyzed with the disclosed methods.
  • references to “a,” “an,” and/or “the” may include one or more than one and that reference to an item in the singular may also include the item in the plural.
  • Reference to an element by the indefinite article “a,” “an” and/or “the” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements.
  • the term “comprise,” and conjugations or any other variation thereof, are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • amplification reaction refers to a method of detecting target nucleic acid by in vitro amplification of DNA or RNA.
  • PCR polymerase chain reaction
  • target or template sequence a specific DNA sequence, termed target or template sequence, that is present in a mixture, by adding two or more short oligonucleotides, also called primers, that are specific for the terminal or outer limits of the template sequence.
  • the template-primers mixture is subjected to repeated Docket No.91482.266WO-PCT cycles of heating to separate (melt) the double-stranded DNA and cooling in the presence of nucleotides and DNA polymerase such that the template sequence is copied at each cycle.
  • primer refers to DNA oligonucleotides complementary to a region of DNA and serves as the initiation of amplification reaction from the 5′ to 3′ direction.
  • a forward and a reverse marker-specific primer can be designed to amplify the marker from a nucleic acid sample.
  • primer pair refers to the forward and reverse primers in an amplification reaction leading to amplification of a double-stranded DNA region of the target.
  • target refers to a nucleic acid region bound by a primer pair that is amplified through an amplification reaction.
  • the PCR “product” or “amplicon” is the amplified nucleic acid resulting from PCR of a set of primer pairs.
  • multiplex amplification reaction herein refers to the detection of more than one template in a mixture by the addition of more than one set of oligonucleotide primers.
  • Amplification is a special case of nucleic acid replication involving template specificity. Amplification may be a template-specific replication or a non-template-specific replication (i.e., replication may be specific template-dependent or not). Template specificity is here distinguished from fidelity of replication (synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid.
  • amplifiable nucleic acid refers to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”
  • PCR product refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
  • PCR generally involves the mixing of a nucleic acid sample, two or more primers or oligonucleotides (primers and oligonucleotides are used interchangeably herein) that are designed to recognize the template DNA, a DNA polymerase, which may be a thermostable DNA polymerase such as Taq or Pfu, and deoxyribose nucleoside triphosphates (dNTP's).
  • a DNA polymerase which may be a thermostable DNA polymerase such as Taq or Pfu
  • dNTP's deoxyribose nucleoside triphosphates
  • the DNA polymerase used can comprise a high fidelity Taq polymerase Docket No.91482.266WO-PCT such that the error rate of incorrect incorporation of dNTPs is less than one per 1,000 base pairs.
  • Reverse transcription PCR quantitative reverse transcription PCR
  • quantitative real time reverse transcription PCR are other specific examples of PCR.
  • the reaction mixture is subjected to temperature cycles comprising a denaturation stage (typically 80-100° C), an annealing stage with a temperature that is selected based on the melting temperature (Tm) of the primers and the degeneracy of the primers, and an extension stage (for example 40-75° C).
  • Detection may comprise contacting the amplified nucleic acid with a probe; and detecting the hybridization of probe with the amplified nucleic acid. Detection may be performed by a variety of methods, such as but not limited to, by a nucleic acid amplification reaction.
  • the amplification reaction maybe an end-point determination or the amplification reaction maybe quantitative.
  • the quantification may be a real-time PCR method.
  • the real-time PCR may be a SYBR® Green Assay or a TAQMAN® Assay.
  • Detection in various embodiments, maybe performed by hybridization using probes specific to target sequences. According to various embodiments, combinations of amplification and hybridization may be used for detection.
  • a marker may be any molecular structure produced by a cell, expressed inside the cell, accessible on the cell surface, or secreted by the cell.
  • a marker may be any protein, carbohydrate, fat, nucleic acid, catalytic site, or any combination of these such as an enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle, or any uni- or multimolecular structure or any other such structure now known or yet to be disclosed whether alone or in combination.
  • a marker may also be called a target and the terms are used interchangeably.
  • a marker may be represented by the sequence of a nucleic acid from which it can be derived or any other chemical structure. Examples of such nucleic acids include miRNA, tRNA, siRNA, mRNA, cDNA, or genomic DNA sequences including complimentary sequences. Alternatively, a marker may be represented by a protein sequence.
  • a marker is not limited to the products of the exact nucleic acid sequence or protein sequence by which it may be represented. Rather, a marker encompasses all molecules that may be detected by a method of assessing the expression of the marker.
  • Expression encompasses any and all processes through which material derived from a nucleic acid template may be produced. Expression thus includes processes such as RNA transcription, mRNA splicing, protein translation, protein folding, post-translational Docket No.91482.266WO-PCT modification, membrane transport, associations with other molecules, addition of carbohydrate moieties to proteins, phosphorylation, protein complex formation and any other process along a continuum that results in biological material derived from genetic material whether in vitro, in vivo, or ex vivo.
  • Expression also encompasses all processes through which the production of material derived from a nucleic acid template may be actively or passively suppressed. Such processes include all aspects of transcriptional and translational regulation. Examples include heterochromatic silencing, transcription factor inhibition, any form of RNAi silencing, microRNA silencing, alternative splicing, protease digestion, posttranslational modification, and alternative protein folding. Expression may be assessed by any number of methods used to detect material derived from a nucleic acid template used currently in the art and yet to be developed.
  • nucleic acid detection methods include any nucleic acid detection method including the following nonlimiting examples, microarray analysis, RNA in situ hybridization, RNAse protection assay, Northern blot, reverse transcriptase PCR, quantitative PCR, quantitative reverse transcriptase PCR, quantitative real-time reverse transcriptase PCR, reverse transcriptase treatment followed by direct sequencing, direct sequencing of genomic DNA, or any other method of detecting a specific nucleic acid now known or yet to be disclosed.
  • library refers to a library of genome/transcriptome-derived sequences.
  • the library may also have sequences allowing amplification of the “library” by the polymerase chain reaction or other in vitro amplification methods well known to those skilled in the art.
  • the library may have sequences that are compatible with next-generation high throughput sequencing platforms.
  • genomic DNA is packed into chromatin as chromosomes within the nucleus.
  • the basic structural unit of eukaryotic native chromatin is the nucleosome, which consists of 146 base pairs (bp) of DNA wrapped around a histone octamer.
  • the histone octamer consists of two copies each of the core histone H2A-H2B dimers and H3-H4 dimers.
  • Nucleosomes are regularly spaced along the DNA in what is commonly referred to as “beads on a string”. Docket No.91482.266WO-PCT The assembly of core histones and DNA into nucleosomes is mediated by chaperone proteins and associated assembly factors.
  • NAP-1 nucleosome assembly protein-1
  • the ATP- independent method for reconstituting chromatin involves the DNA and core histones plus either a protein like NAP-1 or salt to act as a histone chaperone. This method results in a random arrangement of histones on the DNA that does not accurately mimic the native core nucleosome particle in the cell. These particles are often referred to as mononucleosomes because they are not regularly ordered, extended nucleosome arrays and the DNA sequence used is usually not longer than 250 bp (Kundu, T. K. et al., Mol. Cell 6: 551-561, 2000). To generate an extended array of ordered nucleosomes on a greater length of DNA sequence, the chromatin must be assembled through an ATP-dependent process.
  • ATP-dependent assembly of periodic nucleosome arrays which are similar to those seen in native chromatin, requires the DNA sequence, core histone particles, a chaperone protein and ATP-utilizing chromatin assembly factors.
  • ACF ATP-utilizing chromatin assembly and remodeling factor
  • RSF repair and spacing factor
  • the methods of the disclosure can be easily applied to any type of fragmented double stranded DNA crosslinked to chromatin.
  • examples include but are not limited to free DNA isolated from plasma, serum, and/or urine; and apoptotic DNA from cells and/or tissues.
  • the DNA can be fragmented enzymatically in vitro (for example, by DNase I, transposase, and/or restriction endonuclease) or fragmented by mechanical forces (hydro-shear, sonication, nebulization, etc.).
  • Fragmentation of the double stranded DNA can be accomplished by any of the following methods which provide a certain degree over the fragment size: Docket No.91482.266WO-PCT Enzymatic Digestion (Restriction Enzymes): With this method, restriction enzymes, also known as restriction endonucleases, recognize and cut DNA at specific sequences. The cut sites are predictable, allowing for control over the fragment sizes to some extent.
  • Micrococcal Nuclease Digestion Micrococcal nuclease preferentially cuts the DNA at regions that are not protected by proteins, such as nucleosomes. The digestion can be controlled by time and enzyme concentration to achieve the desired fragment size.
  • DNase I Digestion DNase I cuts DNA non-specifically.
  • DNA By adjusting the enzyme concentration and the incubation time, it is possible to partially digest the DNA to obtain fragments of a certain size range.
  • Chemical Cleavage Chemical agents like hydroxyl radicals, copper ions, or other chemical nucleases can be used to cleave DNA. The size of the fragments can be regulated by the concentration of the chemicals and the duration of the exposure.
  • Mechanical Shearing Apart from sonication, DNA can be mechanically sheared through passing it through narrow orifices or needles at high pressure, using devices specifically designed for this purpose.
  • Nebulization DNA is forced through a small hole in a nebulizer device, and the shear forces as the DNA exits the hole create the fragments. The size can be controlled by the pressure and the time of nebulization.
  • Hydrodynamic Shearing DNA is sheared by forces exerted in a fluid flow, such as those created in specific devices like hydro-shear or g-TUBE.
  • Pointed-end Needles or Syringe Pumping Passing DNA solution through narrow- gauge needles or using a syringe pump can create shear forces sufficient to break the DNA into smaller fragments.
  • Laser Fragmentation Short laser pulses can be used to break down DNA into fragments. The energy and frequency of the laser can be controlled to achieve the desired fragment size.
  • Automated Fragmentation Devices There are automated systems like the COVARIS ® system that use adaptive focused acoustics to shear DNA into fragments of controlled size.
  • Methylation of a region in the TERT promoter is shown to be associated with increased transcription.
  • the present method was developed to provide methods of epigenetic editing to measure the impact of manipulating DNA methylation on telomerase activity.
  • the present approach included confirming that DNA methylation is responsible for reactivating telomerase in cancer development.
  • One goal of this approach was to assess molecular mechanisms that are Docket No.91482.266WO-PCT affected by methylation at the TERT promoter.
  • Another goal was to determine whether chromatin conformation and transcription factor (TF) binding are regulated via methylation.
  • Yet another goal was to identify what changes in DNA methylation, TF binding and chromatin conformation are associated with telomerase reactivation in cancer development.
  • FIGS. 1-3 show principles of the present method. Chromatin was prepared from cultured cells by dual crosslinking using formaldehyde and EGS, followed by sonication. Biotinylated PNA probes were hybridized to the telomere sequences, and proteinase K was used to release the telomere-associated sequences.
  • the released DNA sequences were separated from the mixture using streptavidin magnetic beads.
  • the isolated DNA sequences were used to generate the NGS library for high-throughput sequencing.
  • Additional nucleic acid crosslinkers that can be used with the disclosed methods are known to one of skill in the art. See, e.g., Harris and Christian, Meth. Enzymol.468:127-146, 2009. In some examples, a cross linker is light activated.
  • crosslinkers include formaldehyde, disuccinimidyl glutarate, UV-254, psoralens and their derivatives such as aminomethyltrioxsalen, glutaraldehyde, ethylene glycol bis[succinimidylsuccinate], bissulfosuccinimidyl suberate, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) bis[sulfosuccinimidyl] suberate (BS3) and other compounds known to those skilled in the art, including those described in the Thermo Scientific Pierce Crosslinking Technical Handbook.
  • the method involves the use of a peptide nucleic acid (PNA) probe to specifically hybridize DNA sequences involved in chromatin interactions, which are then captured using biotinylated beads.
  • PNA peptide nucleic acid
  • the bead-bound chromatin is then decrosslinked using proteinase K, a serine protease, and the released DNA fragments in the supernatant are purified and subjected to library preparation for next-generation sequencing.
  • sheared chromatin is incubated with 0.25 ⁇ M PNA probe with the following hybridization program: a. 25°C for 3 min b. 71°C for 9 min c. 38°C for 1 h d. 25°C final temperature.
  • Dynabeads MyOne T1 are added to the mix and incubated with probe- hybridized chromatin for 2 hours at room temperature followed by wash steps. After the wash steps, the bead-bound chromatin is resuspended in TE buffer. Proteinase K is added and the Docket No.91482.266WO-PCT mix is incubated at 65°C for 4 hours. The mix is placed on the magnetic stand, and only the supernatant is transferred to a new tube, not the beads. The supernatant is then purified followed by AluI digestion, and regular NGS library preparation is performed.
  • results herein show that the signal resulting from the ligation-free chromatin capture and sequencing method is strongest near chromosome ends and gradually declines over the length of a chromosome arm. Moreover, the resulting signal shows a striking periodicity, suggestive of regularly spaced chromatin loops. The signal is markedly pronounced at sites of interstitial telomeric sequences (ITS), potentially representing chromatin hubs of multiple interacting telomeric loci. Interestingly, a Telomere-C peak was found in the vicinity of the TERT locus, which could point to a telomere-length dependent auto-feedback loop controlling TERT transcription.
  • the disclosed methods can be used to analyze interactions between repeating elements in telomeric sequences and associated segments of genomic DNA, the methods are not limited to this application. In other aspects, the disclosed methods are used to analyze the interactions of transposable elements or interspersed repeats present in a genome with associated segments of genomic DNA. A non-limiting list of such transposable elements or interspersed repeats is available in the Dfam database. In one aspect, the disclosed methods are used to analyze associations between one or more of the transposable elements or interspersed repeats listed in FIG.14 and associated segments of genomic DNA.
  • Retrotransposable Elements are mobile element insertion polymorphisms that are essentially homoplasy-free characters, identical by descent and easy to genotype (reviewed in Batzer M A; Deininger, P L, Alu repeats and human genomic diversity, Nat. Rev. Genet.3(5): 370-9 (2002), doi:10.1038/nrg798).
  • ALUs are REs that are approximately 300 bp insertions and are distributed throughout the human genome in large copy number.
  • REs include smaller families of transposons such as SVA or long interspersed element (“LINE”).
  • SVA elements named after its main components, short interspersed element (“SINE”), variable number tandem repeat (“VNTR”) and Alu element (“ALU”), contain the hallmarks of retrotransposons, in that they are flanked by target site duplications (“TSDs”), terminate in a poly(A) tail and they are occasionally truncated and Docket No.91482.266WO-PCT inverted during their integration into the genome (Ono, M; Kawakami, M; Takezawa, T, A novel human nonviral retroposon derived from an endogenous retrovirus. Nucleic Acids Res. 15(21): 8725-8737 (1987); Wang, H, et al., SVA elements: A hominid-specific retroposon family, J. Mol.
  • telomere-associated complexes are captured by a biotinylated telomeric PNA probe from ⁇ 10 ⁇ g of dual crosslinked chromatin, followed by reverse crosslinking and library preparation. These methods aim to obtain expression data to measure associated changes in transcription.
  • Cell Harvesting and Dual Cross-Linking Chromatin crosslinking is the fundamental step to fix contact information.
  • EGS works for protein-protein crosslinking; while formaldehyde works for protein- DNA crosslinking.
  • over-crosslinking makes chromatin resistant to shearing by sonication.
  • EGS fixative solution in a fume hood by dissolving 0.02 g EGS (Ethylene glycol bis[succinimidylsuccinate]) in 200 ⁇ l DMSO at 37oC for 5 minutes (220mM final concentration).2. Mix 200 ⁇ l EGS/DMSO with 29.8 ml 1X PBS, and then place the EGS mixture at 37 oC until use (1.5mM final concentration).
  • the method performs dual crosslink by adding EGS for crosslink protein-protein interaction preserving better chromatin conformation than single crosslink (i.e., single crosslink refers to using formaldehyde without using EGS).3.
  • Collect cells ( ⁇ 100 million), cultured to ⁇ 80% confluency under recommended conditions, by centrifugation at 200 xg for 5 minutes at room temperature. Discard the medium and wash the cell pellet with 10 ml 1X PBS buffer once. Centrifuge at 200 xg for 5 minutes at Docket No.91482.266WO-PCT room temperature and discard 1X PBS buffer.4. Resuspend cell pellet in 30 ml freshly prepared EGS fixative solution and mix the cell with rotation (10 rpm) for 30 minutes at room temperature.
  • incubation time can be from 20 min to 45 min).5. Add 2 ml 16% (vol/vol) formaldehyde to the fixative solution to a final concentration of 1% (vol/vol) and mix with rotation (10 rpm) for 10 minutes at room temperature. Depending on cell lines, incubation time can be from 5 min to 20 min.6. Quench cross-linking reaction by adding 2.78 ml 2.5M glycine to the fixative solution to a final concentration of 0.2 M with rotation (10 rpm) for 5 min at room temperature.7. Spin down crosslinked cells at 1000 xg for 5min at 4 degrees C.8. Discard quenched media in labeled waste bottle and wash cells twice with 25 mls chilled PBS. 9.
  • D1 Cell Lysis and Sonication Cell Lysis Prepare EB buffer and FA lysis buffer containing 1X proteinase inhibitor.1. Thaw cells on the ice for 5-10 min.2. Resuspend cells in 1 ml of 0.1% FA Lysis buffer with 1X protease inhibitor.3. Rotate at 4°C for 10 min (20rpm).4. Spin at 1700 xg for 5 min at 4°C. Discard the supernatant.5.
  • step 11 the pellet appears puffy and transparent in the final step, and is enough to cover the bottom of the tube. If it's not enough, increase the number of cells for lysis and combine them in the next step. Steps 8-11 permeabilize nuclear to make it easier to be sheared in the next steps.
  • EB buffer wash step is for buffer exchange purpose. Sonication 1. Resuspend pellet with 130 ⁇ l EB buffer.2. Keeping the cap on the COVARIS ® tubes (PN 520045), using a tapered pipette tip, transfer 130 ⁇ l of DNA sample (in EB buffer) by inserting the pipette tip through the pre-split septa. 3. Place COVARIS ® tubes on "Rack 24 Place microTUBE Snap-Cap (PN 500111)".
  • FIG. 9A, 9B and 9C show examples fragment yield and size quality.
  • FIG. 9A shows good quality.
  • FIG.9B shows over shearing.
  • FIG.9C shows under shearing.
  • Chromatin Concentration 1. Carefully transfer the supernatant to Amicon Ultra-0.5ml (Ultracel-100K) column. spin at 4°C 14000 xg for 10 min, discard the bottom part.2. Invert the column to another clean 2 ml collection tube, spin at 4°C 1000 xg for 2 min, to get the concentrated chromatin fragments ( ⁇ 50 ⁇ l).3. This filtered chromatin can be stored at 4 °C overnight.
  • Input chromatin QC (QC2) 1. Take 15 ⁇ l ( ⁇ 3%) of pre-cleared chromatin for QC, and mix with 80 ⁇ l TE and 5 ⁇ l Proteinase K. Incubate in the ThermoMixer at 65°C, 900 rpm for 30-60 min. 2. Purify the sample using Zymo ChIP DNA Clean and Concentrator by following manufacturer's manual, eluted in 30 ⁇ l elution buffer. 3.
  • step 2- 3.5 Keep the lid of the microtube open, and place the tube on a magnetic stand for 2 min and discard the supernatant.4.
  • Repeat step 2- 3.5 Keep the lid of the microtube open, and place the tube on a magnetic stand for 2 min and discard the supernatant.6.
  • Capture QC Take 2 ⁇ l to DNA concentration by Qubit as QC3.
  • the yield of capture DNA should be 1% - 0.1% of input. While, ⁇ 0.1% or undetectable DNA in the no probe control.
  • Docket No.91482.266WO-PCT Secondary Digestion AluI Perform AluI digestion in the End-repairing and A-tailing buffer Input Capture
  • the resulting signal shows a striking periodicity, suggestive of regularly spaced chromatin loops.
  • the Telomere-C signal is markedly pronounced at sites of interstitial telomeric sequences (ITS), potentially representing chromatin hubs of multiple interacting telomeric loci.
  • ITS interstitial telomeric sequences
  • a peak was found in the vicinity of the TERT locus, which could point to a telomere-length dependent auto-feedback loop controlling TERT transcription.
  • FIGS. 4A and 4B show the negative association between resulting signals and chromosome ends.
  • FIG. 4B shows the gradual decline of Telomere-C signal along chromosome arms, indicating that Telomere-chromatin interactions are rich at the end of chromosomes.
  • FIG.4A shows Telomere-C signal (purple bar) and input (blue bar) were visualized using IGV in cell lines at the end of Chr19q13 (CHM13v2).
  • FIG. 4B shows the average Telomere-C signal within 20 MB of the p and q arms was calculated, normalized with input, and displayed in a log scale. The r value of the Pearson correlation and the trend line from the general linear regression model (blue line) are shown.
  • FIGS. 5A, 5B, and 5C show telomeric chromatin interacts in the loci of TERT, CCND2, ITS, and at consensus sites and show cell-specific and consensus Telomere-C signals at different genomic Loc.
  • Telomere-C signal (purple bar) and input (blue bar) were visualized using IGV in the indicated cell lines at the loci of TERT (FIG.5A), CCND 2 (FIG.5B), LINC- 1905 (FIG. 5C), and ITS.
  • Cell-specific signals were observed in TERT and CCND2, while consensus signals were observed in LINC1905 and ITS.
  • FIGS.6A-6D show the periodicity of Telomere-C signals at the end of chromosomes.
  • FIGS.6A and 6B show Telomere-C signal within 20 MB of the p arm in BJ (FIG.6A) or (in FIG.6B) A2780 cells, calculated and normalized with input and displayed in a log scale.
  • FIGS. 6C and 6D display periodograms, showing the dominant and minor frequency of Telomere-C signal.
  • the periodicity of the Telomere-C signal suggests long-distance telomere-chromatin interactions.
  • FIGS. 7A, 7B, and 7C show enrichment of Telomere-C signals in ITS regions.
  • FIG. 7A shows a heatmap showing Telomere-C signals in the ITS region and its upstream and downstream regions in A2780, BJ, IMR90, and WI38 cells.
  • FIG.7C is an ideogram to show the distribution of ITS in 45 mappable (blue) or 33 unmappable regions (black). Telomere-C signal is markedly pronounced at interstitial telomeric sequences (ITS) sites, indicating that ITS serves as a hub of telomere-chromatin interaction.
  • ITS interstitial telomeric sequences
  • telomere-chromatin interaction in the terminal regions of chromosomes (proximal), regions of interstitial telomere sequences (ITSs), or centromeric ITSs (distal) in chromosome 20 can be easily visualized in these representative results.
  • FIGS. 11A-11C the richness of Telomere-C signals in the Telomere Associated Repeat 1, (CCCTAA)n, or (TTAGGG)n, and other repeat elements is shown.
  • the signal of Telomere-C in the regions of repeat elements has been analyzed by comparing the signals between repeat regions and random regions.
  • Telomere-C is rich in the non-telomeric (TTAGGG)n or (CCCTAA)n regions, which are potential regions of ITS. Additionally, Telomere-C signals have been found to be Docket No.91482.266WO-PCT significantly rich in repeat elements that don’t contain TTAGGG (or CCCTAA) sequences such as telomere-associated repeats (TAR1) or D20S16. The same discovery has been found in early passages and later passages of BJ cells (BJP14 and BJP22), and IMR90 cells. Validation of Telomere-ITSs interaction by 3C is shown in FIGS. 12A and 12B.
  • Chromatin conformation capture (3C) was performed to detect the interaction between telomere and selected ITSs on chromosome 18 or chromosome 19. Chromatin was crosslinked with 1.5mM EGS and 1% formaldehyde followed by enzymatic digestion with Alu I. Proximity ligation with T4 ligase (or without T4 ligase as negative control) was conducted, and reverse crosslinking was performed with proteinase K. The 3C DNA was purified applying the phenol-chloroform extraction method for the PCR reaction with customized primers. Electrophoresis in 1% agarose gel was used to visualize the results.
  • Tel1-F (GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT) (SEQ ID NO: 1) and ITS18-RC (ATGGGTAAAGGTCAGGGTCACG) (SEQ ID NO: 2) or ITS19-RC (GGTGACGCCCTGTATGCAAA) (SEQ ID NO: 3) were used to detect ligated molecules containing telomere and ITS sequence in the PCR reaction.
  • the electrophoresis result shows a smear in ligated 3C DNA, but not in non-ligated 3C DNA. This supports the hypothesis since the Tel1-F primer can bind to any loci of telomere resulting vary sizes of PCR product.
  • ITS19-F TTTTGGGTATCATGTGTGCATTAGG
  • ITS19-R AGCCCCGTCTTGCAGTCTTT
  • ITS18-F GCTGATCAGGACGCTTTTGC
  • ITS18-R TCCTAACGAGGTTCTCCCCA
  • telomeres or subtelomeres
  • ITS Fluorescent in situ hybridization
  • Cells in the interphase were fixed and hybridized with a PNA probe covering telomere regions and a DNA oligo probe covering a selected ITS region (or subtelomere, p-arm) on chromosome 18.
  • the nucleus was imaged, the distance between two foci was measured, and it was determined whether they were adjacent ( ⁇ 0.5 ⁇ m). It was found that over 50% of the foci between the telomere and ITS are adjacent, while more than 75% of the foci between subtelomere and ITS are separated.
  • the data suggests an interaction between telomere and ITS in a trans manner.
  • references to “a,” “an,” and/or “the” may include one or more than one and that reference to an item in the singular may also include the item in the plural.
  • Reference to an element by the indefinite article “a,” “an” and/or “the” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements.
  • the term “comprise,” and conjugations or any other variation thereof, are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

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Abstract

L'invention propose des procédés de capture de loci d'ADN associés à des télomères à partir de la chromatine. La chromatine a été préparée à partir de cellules cultivées par double réticulation à l'aide de formaldéhyde et d'EGS, suivie d'une sonication. Des sondes PNA biotinylées ont été hybridées aux séquences d'ADN télomère, et une sérine protéase a été utilisée pour libérer les séquences d'ADN associées aux télomères. Les fragments d'ADN libérés dans le surnageant ont été purifiés et soumis à une préparation de bibliothèque pour le séquençage de nouvelle génération. Le procédé de capture et de séquençage de chromatine sans ligature enrichit des loci d'ADN associés à des télomères spécifiques pour fournir une représentation précise de l'interaction de chromatine. Des aperçus dans les interactions de chromatine se produisant au niveau de télomères sont utilisés pour étudier le rôle potentiel de télomères dans le vieillissement et les maladies liées à l'âge.
PCT/US2024/027099 2023-04-30 2024-04-30 Procédés de capture de conformation de chromatine sans ligature avec séquençage à haut débit Pending WO2024229044A1 (fr)

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US20140024052A1 (en) * 2010-11-10 2014-01-23 The Trustees Of Princeton University Sequence-specific extraction and analysis of dna-bound proteins

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US20140024052A1 (en) * 2010-11-10 2014-01-23 The Trustees Of Princeton University Sequence-specific extraction and analysis of dna-bound proteins

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Title
ZENG PING-YAO, VAKOC CHRISTOPHER R., CHEN ZHU-CHU, BLOBEL GERD A., BERGER SHELLEY L.: "In Vivo Dual Cross-Linking for Identification of Indirect DNA-Associated Proteins by Chromatin Immunoprecipitation", BIOTECHNIQUES, INFORMA HEALTHCARE, US, vol. 41, no. 6, 1 December 2006 (2006-12-01), US , pages 694 - 698, XP093233432, ISSN: 0736-6205, DOI: 10.2144/000112297 *

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