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EP1381692A2 - Emplacement et fonction de proteines de liaison a l'adn a l'echelle du genome - Google Patents

Emplacement et fonction de proteines de liaison a l'adn a l'echelle du genome

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Publication number
EP1381692A2
EP1381692A2 EP01994453A EP01994453A EP1381692A2 EP 1381692 A2 EP1381692 A2 EP 1381692A2 EP 01994453 A EP01994453 A EP 01994453A EP 01994453 A EP01994453 A EP 01994453A EP 1381692 A2 EP1381692 A2 EP 1381692A2
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EP
European Patent Office
Prior art keywords
dna
cell
ofthe
protein
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01994453A
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German (de)
English (en)
Inventor
John Wyrick
Richard A. Young
Bing Ren
François Robert
Itamar Simon
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Whitehead Institute for Biomedical Research
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Whitehead Institute for Biomedical Research
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Publication of EP1381692A2 publication Critical patent/EP1381692A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • Transcriptional activators for example, bind to specific promoter sequences and recruit chromatin modifying complexes and the transcription apparatus to initiate RNA synthesis.
  • the remodeling of gene expression that occurs as cells move through the cell cycle, or when cells sense changes in their environment, is effected in part by changes in the DNA-binding status of transcriptional activators.
  • Distinct DNA-binding proteins are also associated with centromeres, telomeres, and origins of DNA replication, where they regulate chromosome replication and maintenance.
  • CDK cyclin-dependent
  • Proteins which bind to a particular region of DNA can be detected using known methods. However, a need exists for a method which allows examination of the binding of proteins to DNA across the entire genome of an organism.
  • the present invention relates to a method of identifying a region (one or more) of a genome of a cell to which a protein of interest binds.
  • DNA binding protein of a cell is linked (e.g., covalently crosslinked) to genomic DNA of a cell.
  • the genomic DNA to which the DNA binding protein is linked is identified and combined or contacted with DNA comprising a sequence complementary to genomic DNA ofthe cell (e.g., all or a portion of a cell's genomic DNA such as one or more chromosome or chromosome region) under conditions in which hybridization between the identified genomic DNA and the sequence complementary to genomic DNA occurs.
  • Region(s) of hybridization are region(s) ofthe genome ofthe cell to which the protein of interest binds.
  • proteins which bind DNA in a cell are crosslinked to the cellular DNA.
  • the resulting mixture which includes DNA bound by protein and DNA which is not bound by protein is subject to shearing conditions.
  • DNA fragments ofthe genome crosslinked to DNA binding protein are generated and the DNA fragment (one or more) to which the protein of interest is bound is removed from the mixture.
  • the resulting DNA fragment is then separated from the protein of interest and amplified, using known methods.
  • the DNA fragment is combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragment and a region ofthe sequence complementary to genomic DNA occurs; and the region of the sequence complementary to genomic DNA to which the DNA fragment hybridizes is identified.
  • the identified region (one or more) is a region ofthe genome ofthe cell, such as a selected chromosome or chromosomes, to which the protein of interest binds.
  • the present invention relates to a method of identifying a region of a genome (such as a region of a chromosome) of a cell (test sample) to which a protein of interest binds, wherein the DNA binding protein ofthe cell is crosslinked to genomic DNA ofthe cell using formaldehyde.
  • DNA fragments ofthe crosslinked genome are generated and the DNA fragment to which the protein of interest is bound is removed or separated from the mixture, such as through immunoprecipitation using an antibody that specifically binds the protein of interest. This results in separation ofthe DNA-protein complex.
  • the DNA fragment in the complex is separated from the protein of interest, for example, by subjecting the complex to conditions which reverse the crosslinks.
  • the separated DNA fragment is amplified (e.g., non-specifically) using ligation-mediated polymerase chain reaction (LM-PCR), and then fluorescently labeled.
  • the labeled DNA fragment is contacted with a DNA microarray comprising a sequence complementary to genomic DNA of the cell, under conditions in which hybridization between the DNA fragment and a region ofthe sequence complementary to genomic DNA occurs.
  • the region ofthe sequence complementary to genomic DNA to which the DNA fragment hybridizes is identified by measuring fluorescence intensity, and the fluorescence intensity ofthe region ofthe sequence complementary to genomic DNA to which the DNA fragment hybridizes is compared to the fluorescence intensity of a control. Fluorescence intensity in a region ofthe sequence complementary to genomic DNA which is greater than the fluorescence intensity ofthe control in that region ofthe sequence complementary to genomic DNA marks the region ofthe genome in the cell to which the protein of interest binds.
  • Also encompassed by the present invention is a method of determining a function of a protein of interest which binds to the genomic DNA of a cell.
  • DNA binding protein ofthe cell is crosslinked to the genomic DNA ofthe cell.
  • DNA fragments ofthe genome crosslinked to DNA binding protein are then generated, as described above, and the DNA fragment (one or more) to which the protein of interest is bound is removed from the mixture.
  • the resulting DNA fragment is then separated from the protein of interest and amplified.
  • the DNA fragment is combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragment and a region ofthe sequence complementary to genomic DNA occurs; and the region of the sequence complementary to genomic DNA to which the DNA fragment hybridizes is identified.
  • This identified region is a region ofthe genome of the cell to which the protein of interest binds.
  • the identified region is characterized and the characteristic ofthe identified region indicates the function ofthe protein of interest (e.g., a regulatory protein such as a transcription factor; an oncoprotein).
  • the present invention also relates to a method of determining whether a protein of interest which binds to genomic DNA of a cell functions as a transcription factor.
  • DNA binding protein ofthe cell is crosslinked to the genomic DNA ofthe cell. DNA fragments ofthe crosslinked genome are generated and the DNA fragment to which the protein of interest is bound is removed from the mixture. The resulting DNA fragment is separated from the protein of interest and amplified.
  • the DNA fragment is combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragment and a region ofthe sequence complementary to genomic DNA occurs.
  • the region ofthe sequence complementary to genomic DNA to which the DNA fragments hybridizes is identified; wherein if the region ofthe genome is a regulatory region, then the protein of interest is a transcription factor.
  • the present invention also relates to a method of identifying a set of genes, the members of which are genes for which cell cycle regulator binding correlates with gene expression.
  • the method comprises identifying a set of genes that is bound in vivo by at least one cell cycle regulator (e.g., transcriptional activator) in a selected cell type (e.g., mammalian cell, yeast cell); comparing the set of genes identified with genes whose expression levels vary in a periodic manner during the cell cycle ofthe selected cell type; and identifying genes that are bound by one or more ofthe cell cycle regulators, thus identifying a set of genes, the members of which are genes whose expression levels vary in a periodic manner during the cell cycle and are bound by at least one cell cycle regulator, wherein the set identified is referred to as a set of genes, the members of which are genes for which cell cycle regulator binding correlates with gene expression.
  • a cell cycle regulator e.g., transcriptional activator
  • the methods described herein facilitate the dissection ofthe cells regulatory network of gene expression across the entire genome and aid in the identification of gene function.
  • Work described herein provides the basis for constructing a complete map ofthe transcriptional regulatory network that controls the cell cycle. In one embodiment, it forms the foundation for a complete map ofthe transcriptional regulatory network that controls the yeast cell cycle.
  • Figure 1 is an illustration ofthe Genome-wide Monitoring Protein-DNA interactions described herein.
  • Figure 2 shows how the relative binding ofthe protein of interest to each sequence represented on an array was calculated using a weighted average analysis.
  • Figure 3 is a graph of chromosomal position versus fold change of Genome- wide Monitoring Protein-DNA interactions.
  • Figure 4 is a graph of chromosome position versus ratio of tagged to untagged for binding of ORC1 to yeast chromosome HI.
  • Figure 5 A is an example of a scanned image.
  • the unenriched and IP enriched DNA generates green fluorescence and red fluorescence respectively.
  • the close-up image shows examples of spots for which the red intensity is over- represented, indicating binding ofthe targeted protein to these DNA sequences.
  • Figure 5B show that small amounts of DNA can be quantitatively amplified and labeled with Cy3 and Cy5 fluorophores.
  • Cy3- and Cy5-labeled DNA from 1 ng of yeast genomic DNA was prepared using the LM-PCR method described in the text. The resulting DNA samples were mixed and hybridized to a yeast intragenic DNA microarray. Low intensity spots have larger variations than high intensity spots, probably due to background noise.
  • Figure 6A shows the set of 24 genes whose promoter regions are most likely to be bound by Gal4 by the analysis criteria described herein.
  • Figure 6B is a schematic ofthe Gal4 binding intergenic regions.
  • Figure 6C shows the results of conventional ChlP analysis.
  • Figure 6D shows the results ofthe AlignAce program used to identify a consensus binding site for the Gal4 activator.
  • Figure 6E is a bar graph showing relative expression of PLC10 and MTHl .
  • Figure 6F is a schematic illustrating how the identification of MTHl and MTH, PCL10 and FUR4 as Gal4-regulated genes reveals how several different metabolic pathways are interconnected.
  • Figure 6G contains three graphs showing galactose-induced expression of FUR4, MTHl and PLC10 is GAL4-dependent; samples from wild-type and gal4- strains were taken before and after addition of galactose.
  • MTH1 and PLC10 was monitored by quantitative reverse transcriptase-PCR (RT- PCR) ans was quantified by phosphoimaging.
  • Figure 7 lists the set of genes whose promoter regions are most likely to be bound by Stel 12 by the analysis criteria described herein.
  • Figure 8 is a schematic of a model summarizing the role of Stel 2 target genes in the yeast mating pathway. Gray boxes denote the cellular processes known to be involved in mating; yellow boxes denote cellular processes that are likely associated with mating. Genes in black were previously reported to be associated with the mating process; genes in red are Stel 2 targets that likely play a role in mating.
  • Figures 9A-9C show the cell cycle transcriptional regulators study design.
  • Figure 9A depicts the stages ofthe cell cycle together with yeast cell morphology (brown) and transcriptional regulators (blue); the transcriptional regulators are positioned at the stage during which they have been reported to function (Breedon et al, Curr. Biol, 70.R586-R588(2000), Mendenhall et al, Mol Biol. Rev., 67. 1191-1243 (1998)).
  • Figure 9B is a scatter plot of Cy5 versus Cy3 intensities for a control experiment in which aliquots of whole cell extract (WCE) were independently labeled with Cy3 and Cy5 and hybridized to a DNA microarray containing all yeast intergenic regions.
  • WCE whole cell extract
  • Figure 9C is a scatter plot of an experiments in which the Fkh2 IP-enriched DNA was labeled with Cy5 and the WCE was labeled with Cy3.
  • the red and blue lines border the regions with confidence levels of pO.OOl and pO.Ol, respectively.
  • the cpols whose values have confidence levels of pO.OOl represent promoters most likely bound by the Fkh2 factor.
  • Figures 10A-10B show genome-wide location ofthe nine cell cycle transcription factors.
  • Figure 10A show the 213 ofthe 800 cell cycle genes whose promoter regions were bound by a myc-tagged version of at least one ofthe nine cell cycle transcription factors (p ⁇ 0.001) are represented as horizontal lines.
  • FIG. 1 OB is a schematic in which the circle represents a smoothed distribution ofthe transcription timing (phase) ofthe 800 cell cycle genes (Spellman et al, Mol. Cell Biol Cell, P/3273-3297 (1998)).
  • the intensity ofthe red color normalized by the maximum intensity value for each factor, represents the fraction of genes expressed at that point that are bound by a specific activator.
  • the similarity in the distribution of color for specific factors shows that these factors bind to genes that are expressed during the same time frame.
  • Figures 11 A-l IB are schematics showing transcriptional regulation of cell cycle transcription factor genes.
  • Figure 11 A shows a summary of previous evidence for regulation of cell cycle transcription factor genes and CLN3 transcriptional regulators (Althoefer et al, Mol. Cell Biol, /J/5917-5928 (1998); Foster et al. Mol. Cell Biol, 13:3792- 3801 (1993); Koranda et al, Nature, 406:94-98 (2000); Kumar et al. Curr. Biol, 10:896-906 (2000); Kuo et al, Mol. Cell Biol. ,14:3348-359 (1994); Loy et al Mol.
  • Figure 1 IB is a model for the closed regulatory circuit produced by cell cycle transcriptional regulators based on genome-wide binding data.
  • the genome-wide location data indicate that each group of transcriptional activators regulates activators acting in the next cell cycle stage.
  • the red arrows represent binding of a transcription factor to the promoter of another regulatory factor.
  • the blue arrows represent posttranslational regulation.
  • Figures 12A-12B are schematics showing transcriptional regulation of cyclin and cyclin/CDK regulator genes.
  • Figure 12A shows a summary of previous evidence for transcriptional regulation of genes encoding the cyclins (green) and cyclin CDK regulators (red) by the cell cycle transcription factors (Althoefer et al. Mol. Cell Biol, 15:5917-5928 (1998); Dirick et al. Nature, 557/508-513 (1992); Hollenhorst et al. Genetics, 75 1533-1548 (2000); Iyer et al. Nature, 409:533-536 (2001); Knapp et al. Mol. Cell Biol, 76/5701-5707 (1998); Koch et al.
  • Figure 12B is a model for transcriptional regulation of cyclin and cyclin CDK regulators based on previous studies and on genome-wide binding data. Each group of transcription factors regulates key cell cycle regulators that are needed for progression through the cell cycle.
  • Figure 13 is a schematic ofthe regulation of cell cycle functions by the activators. Stage-specific cell cycle functions under the control of specific factors are shown.
  • the budding category include genes involved in budding and in cell wall biogenesis;
  • the DNA replication category includes genes involved in replication, /059371
  • the chromatin category includes genes encoding histones, chromatin modifiers, and telomere length regulators. The identity and functions of genes in each category are listed in Table 3.
  • Figures 14A-14C are diagrams showing partial redundancy between homologous activators.
  • Figure 14A are Venn diagrams depicting the overlap between the targets of pairs of homologous cell cycle transcriptional regulatory proteins.
  • the numbers in parenthesis under each activator represent the sum of cell cycle genes whose promoters were bound by the protein.
  • the number in the intersection between two circles reflects the numbers of genes whose promoters were bound by both proteins.
  • Figure 14B are Venn diagrams representing the overlap in target sites between pairs of regulatory proteins that reside within the same complex.
  • Figure 14C is a Verm diagram representing the overlap in target sites between two transcriptional regulators that are not known to be related.
  • telomeres a genome-wide location profiling method for DNA-bound proteins, which has been used to monitor dynamic binding of gene-specific transcription factors and components ofthe general transcription apparatus in yeast cells.
  • the genome-wide location method correctly identified known sites of action for the transcriptional activators Gal4 and Stel2 and revealed unexpected functions for these activators.
  • the combination of expression and location profiles identified the global set of genes whose expression is under the direct control of specific activators and components ofthe transcription apparatus as cells responded to changes in their extracellular environment. Genome-wide location analysis provides a powerful tool for further dissecting gene regulatory networks, annotating gene functions and exploring how genomes are replicated. 059371
  • the present invention provides methods of examining the binding of proteins to DNA across the genome (e.g., the entire genome or a portion thereof, such as one or more chromosomes or a chromosome regions) of an organism.
  • the present invention relates to a method of identifying a region (one or more) of genomic DNA of a cell to which a protein of interest binds.
  • proteins which bind DNA in a cell are crosslinked to the cellular DNA.
  • the resulting mixture which includes DNA bound by protein and DNA which is not bound by protein is subject to shearing conditions.
  • DNA fragments ofthe genome crosslinked to DNA binding protein are generated and the DNA fragment (one or more) to which the protein of interest is bound is removed from the mixture.
  • the resulting DNA fragments are then separated from the protein of interest and amplified using known techniques.
  • the DNA fragment is then combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragments and the sequence complementary to genomic DNA occurs; and the region ofthe sequence complementary to genomic DNA to which the DNA fragment hybridizes is identified.
  • the identified region is a region ofthe genome ofthe cell to which the protein of interest binds.
  • Also encompassed by the present invention is a method of determining a function of a protein of interest which binds to the genomic DNA of a cell.
  • DNA binding protein ofthe cell is crosslinked to the genomic DNA ofthe cell.
  • DNA fragments ofthe genome crosslinked to DNA binding protein are then generated, as described above, and the DNA fragment (one or more) to which the protein of interest is bound is removed.
  • the resulting DNA fragment is then separated from the protein of interest and amplified.
  • the DNA fragment is then combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragment and a region ofthe sequence complementary to genomic DNA occurs; and the region of the sequence complementary to genomic DNA to which the DNA fragment hybridizes is identified and is a region ofthe genome ofthe cell to which the protein of interest binds.
  • the identified region is characterized (e.g., a regulatory region) 059371
  • the characteristic ofthe identified region indicates a function ofthe protein of interest (e.g., a transcription factor; an oncoprotein).
  • a function ofthe protein of interest e.g., a transcription factor; an oncoprotein.
  • the present invention also relates to a method of determining whether a protein of interest which binds to genomic DNA of a cell functions as a transcription factor.
  • DNA binding protein ofthe cell is crosslinked to genomic DNA ofthe cell and DNA fragments ofthe crosslinked genome are generated.
  • the DNA fragment to which the protein of interest is bound are removed.
  • the resulting DNA fragment is separated from the protein of interest and amplified.
  • the DNA fragment is combined with DNA comprising a sequence complementary to genomic DNA ofthe cell, under conditions in which hybridization between the DNA fragments and sequence complementary to genomic DNA occurs.
  • the region ofthe sequence complementary to genomic DNA to which the DNA fragments hybridizes is identified wherein if the region ofthe genome is a regulatory region, then the protein of interest is a transcription factor.
  • the methods ofthe present invention can be used to examine and/or identify
  • DNA binding of proteins across the entire genome of a eukaryotic organism For example, DNA binding proteins across the entire genome of eukaryotic organisms such as yeast, Drosophila and humans can be analyzed. Alternatively, they can be used to examine and or identify DNA binding of proteins to an entire chromosome or set of chromosomes of interest.
  • genome-wide location analysis has been used to identify the in vivo genome binding sites for cell cycle transcription factors, in particular genome binding sites for each ofthe known yeast cell cycle transcription factors.
  • Such analysis is useful to identify genome binding sites (genomic targets) of cell cycle regulators (transcriptional activators) in a variety of cell types and, as also described herein, has resulted in identification of genomic targets of each ofthe nine known yeast cell cycle transcription activators.
  • One embodiment ofthe present invention is a method of identifying genes that are expressed in a periodic manner during the cell cycle of a selected cell type and are bound by a cell cycle regulator(s) or cell cycle transcription factors, also referred to transcription(al) regulators/activators.
  • the method is, thus, one of identifying a set of genes where cell cycle factor binding correlates with gene expression.
  • a set of genes whose factor binding correlates with gene expression at a selected level of stringency of the analysis criteria for binding data is identified.
  • the stringency ofthe analysis criteria for binding data can be p ⁇ 0.001, pO.Ol, p ⁇ 0.05 or another selected level and preferably will be selected at such a level that few or no false positives are detected.
  • Cell cycle regulators can be identified by the method of the present invention in a wide variety of cell types (referred to as selected cell types, such as eukaryotic (mammalian, nonmammalian) cells, including human and nonhuman cells (including, but not limited to, yeast and other fungi, worm, fly, avian, murine, canine, bovine, feline, equine, and nonhuman primate cells).
  • selected cell types such as eukaryotic (mammalian, nonmammalian) cells, including human and nonhuman cells (including, but not limited to, yeast and other fungi, worm, fly, avian, murine, canine, bovine, feline, equine, and nonhuman primate cells).
  • the method is carried out, in one embodiment, by identifying a set of genes that is bound in vivo by a cell cycle regulator(s) or transciption factor(s) in a selected cell type (e.g., from a particular organism, which can be human or nonhuman, such as those listed above); comparing that set of genes with genes whose expression levels vary in a periodic manner during the cell cycle of that organism; and identifying genes that are bound by one or more ofthe cell cycle regulators (identifying genes whose factor binding correlates with gene expression), thus identifying genes whose expression levels vary in a periodic manner during the cell cycle and are bound a cell cycle factor(s). Genes identified in this manner can be characterized, as described herein.
  • a set of yeast genes for which factor binding correlates with gene expression has been identified by comparing the set of genes bound by the nine cell cycle transcription factors with the approximately 800 genes whose expression levels vary in a periodic fashion during the yeast cell cycle.
  • Results of work described herein generally support the model for stage- specific regulation of gene expression, described by others, by these activators and extend it to encompass promoters for several hundred cell cycle genes; confirmed results of earlier studies, which established that genes encoding several ofthe cell cycle transcriptional regulators are themselves bound by other cell cycle functions; revealed that cell cycle transcriptional control is effected by a connected regulatory network of transcriptional activators; and identified a set of promoters bound in vivo by each ofthe cell cycle regulators, which were further analyzed and shown to comprise consensus binding sequence motifs (see Table 2).
  • a variety of proteins which bind to DNA can be analyzed.
  • any protein involved in DNA replication such as a transcription factor, or an oncoprotein can be examined in the methods ofthe present invention.
  • methods which can be used to link DNA binding protein ofthe cell to the genome ofthe cell For example, UV light can be used.
  • formaldehyde is used to crosslink DNA binding proteins to the genomic DNA of a cell.
  • identification of DNA fragments bound to the protein of interest can be removed from the mixture comprising DNA fragment(s) bound to the protein of interest and DNA fragments which are not bound to the protein of interest, using a variety of methods.
  • immunoprecipitation using an antibody e.g., polyclonal, monoclonal
  • an antibody e.g., polyclonal, monoclonal
  • antigen binding fragment thereof which binds (specifically) to the protein of interest
  • the protein of interest can be labeled or tagged using, for example, an antibody epitope (e.g., hemagglutinin (HA)).
  • HA hemagglutinin
  • the DNA fragments in the methods described herein can be amplified using any suitable method.
  • the DNA is amplified using a non-specific amplification method.
  • a non-specific amplification method For example, ligation-mediated polymerase chain reaction (e.g., see Current Protocols in Molecular Biology, Ausubel, F.M. et al, eds. 1991, the teachings of which are incorporated herein by reference) can be used.
  • the present invention provides a method for non-specifically amplifying DNA fragments from the entire genome of a cell.
  • non-specific amplification can be used without increasing the signal-to-noise ratio.
  • the ability to non-specifically amplify DNA fragments from an entire genome of a cell constitutes a important distinction over other techniques, such as the ChlP technique which relies upon specific primer-based amplification.
  • the amplified DNA can be labeled (e.g., a radioactive label, a non-radioactive label such as a fluorescent label) to facilitate identification.
  • the DNA is labeled using a fluorescent dye, such as Cy5 or Cy3.
  • the DNA comprising the complement sequence ofthe genome ofthe cell can be combined with the isolated DNA fragment to which the protein of interest binds using a variety of methods.
  • the complement sequence can be immobilized on a glass slide (e.g., microarray such as the Corning Microarray Technology (CMTTM) GAPSTM) or on a microchip.
  • a glass slide is used which can accommodate an entire genome of a cell (e.g., at least about 7200 spots (DNA)).
  • Conditions of hybridization used in the methods ofthe present invention include, for example, high stringency conditions and/or moderate stringency conditions. See e.g., pages 2.10.1-2.10.16 (see particularly 2.10.8-11 ) and pages 6.3.1 -6 in Current Protocols in Molecular Biology). Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of hybridization. Thus, high or moderate stringency conditions can be determined empirically, and depend in part upon the characteristics ofthe known nucleic acids (DNA, RNA) and the other nucleic acids to be assessed for hybridization thereto.
  • the methods ofthe present invention can further comprise comparing the results to a control (control sample).
  • a control control sample
  • the methods ofthe present invention can be carried out using a control protein which is not a DNA binding protein.
  • immunoprecipitation is performed using an antibody against an HA or MYC epitope tag. The results of immunoprecipitating the protein of interest containing the tag, and the protein of interest without the tag are compared. The untagged protein should not be immunoprecipitated, and thus, serves as a negative control.
  • Using the methods ofthe present invention also provides for the ability to compare the sample with the control sample simultaneously.
  • a test sample if hybridized to an array and compared to a control sample which has been hybridized to a different array and a ratios is cal culated to determine binding results.
  • two samples e.g., a test sample and a control sample
  • two anays e.g., an array for the test sample and another array for the control sample.
  • the difference between arrays due to manufacturing artifacts is a major source of noise, which can be eliminated using the methods described herein.
  • a particular embodiment ofthe present invention comprises the combined use of Chromatin Immunoprecipitation (ChIP) and Genome-wide expression monitoring microarrays.
  • Chromatin immunoprecipitation allows the detection of proteins that are bound to a particular region of DNA. It involves four steps: (1) formaldehyde cross-linking proteins to DNA in living cells, (2) disrupting and then sonicating the cells to yield small fragments of cross-linked DNA, (3) immunoprecipitating the protein-DNA crosslinks using an antibody which specifically binds the protein of interest, and (4) reversing the crosslinks and amplifying the DNA region of interest using the
  • PCR Polymerase Chain Reaction
  • the present method is not limited to amplifying individual DNA regions by performing PCR with specific primers. Rather the entire genome (test sample) is amplified (e.g., non-specifically) using a Ligation-mediated PCR (LMPCR) strategy.
  • the amplified DNA was fluorescently labeled by including fluorescently- tagged nucleotides in the LM-PCR reaction.
  • the labeled DNA was hybridized to a DNA microarray containing spots representing all or a subset (e.g., a chromosome or chromosomes) ofthe genome.
  • the fluorescent intensity of each spot on the microarray relative to a non-immunoprecipitated control demonstrated whether the protein of interest bound to the DNA region located at that particular spot.
  • the methods described herein allow the detection of protein- DNA interactions across the entire genome.
  • DNA microarrays consisting of most of yeast chromosome HI plus approximately 15 model genes whose expression have been well studied were constructed. These arrays were used in conjunction with the ChIP technique to study the DNA-binding properties of transcription factors and the transcription apparatus genome-wide.
  • the methods described herein provide insights into the mechanism and regulation of gene expression in eukaryotic cells.
  • the genome-wide location analysis method described herein allows protein- DNA interactions to be monitored across the entire yeast genome and is diagramed in Figure 1.
  • the method combines a modified Chromatin Immunoprecipitation (ChIP) procedure, which has been previously used to study in vivo protein-DNA interactions at one or a small number of specific DNA sites, with DNA microarray analysis. Briefly, cells are fixed with formaldehyde, harvested by sonication, and DNA fragments that are crosslinked to a protein of interest are enriched by immunoprecipitation with a specific antibody. After reversal ofthe crosslinking, the enriched DNA is amplified and labeled with a fluorescent dye (e.g., Cy5) using ligation-mediated PCR (LM-PCR).
  • LM-PCR ligation-mediated PCR
  • a sample of DNA that has not been enriched by immunoprecipitation is subjected to LM-PCR in the presence of a different fluorophore (e.g., Cy3), and both immunoprecipitation (IP)-enriched and unenriched pools of labeled-DNA are hybridized to a single DNA microarray containing all yeast intergenic sequences.
  • a different fluorophore e.g., Cy3
  • IP immunoprecipitation
  • X (a 2 - a,)/[ ⁇ , 2 + ⁇ 2 2 + f 2 (a, 2 + where a 1>2 are the intensities measured in the two channels for each spot, ⁇ , 2 are the uncertainties due to background subtraction, and f is a fractional multiplicative error such as would come from hybridization non-uniformities, fluctuations in the dye incorporation efficiency, scanner gain fluctuations, ets.
  • X is approximately normal.
  • the parameters ⁇ and f were chosen such that X has unit variance. The significance of a change of magnitude
  • is then calculated as
  • the data for the intensity of each spot on an array, as well as the intensity and standard deviation around each spot is measured; and this is calculated for both the test sample and the control sample hybridized on the same array.
  • These measurements are used to calculate the enrichment in a probabilistic fashion using a mathematical model.
  • each measurement is weighed allowing replicates to be combined appropriately which addresses the susceptibility of spots with lower signals to generate more noise.
  • MODEL GENES ARRAY FOR THE CHARACTERIZATION OF PROTEIN-DNA INTERACTIONS Array contains all non-overlapping open reading frames (ORF) on
  • Chromosome IH (See Table 1). When a sequence contains part or all of two potential reading frames, the larger sequence was chosen to represent the ORF. Any remaining sequence was included in intergenic fragments.
  • Saccharomyces Genomic Database SGD functional chromosomal map.
  • Reactions were quantified by visualizing l ⁇ l ofthe purified DNA on an agarose gel compared to a known quantity of lambda DNA cut with Hindrfl (Promega).
  • DNA was stored at -20 until shortly before printing. The DNA was then dried down by speed vac in the Coming microtiter plates to less than 5 ⁇ l.
  • PCR reactions were resuspended to approximately 0.5 mg/ml in 3XSSC.
  • SSC was made as a 20X stock (3M NaCl, 0.3M Na 3 citrate-2H 2 O, pH'd to 7.0 with HCI) and diluted to the desired concentration with H 2 O.
  • PCR products should be greater than 250 pb.
  • the probe volume should be 20-30 ⁇ l for a small coverslip (25 mm 2 ) and 40- 60 ⁇ l for a large cover slip (24 x 60 mm).
  • Gal4 was selected because it is among the best characterized transcriptional activators, it is known to be responsible for induction of genes necessary for galactose metabolism, and a consensus DNA binding sequence (the UAS G ) has been identified for Gal4 in the promoters ofthe GAL genes. Very little Gal4 is bound at the UAS G ofthe GAL1 and GAL10 promoters when cells are grown in glucose (the repressed state), whereas relatively high levels of Gal 4 are bound in galactose (the activated state).
  • the genome- wide location of epitope-tagged Gal4p in both glucose and galactose media was investigated in three independent experiments, as described in more detail below.
  • the location analysis experiment identified seven genes previously reported to be regulated by Gal 4 and three additional genes encoding activities that are physiologically relevant to cells that utilize galactose as the sole carbon source, but which were not previously known to be regulated by this activator ( Figures 6A).
  • MTH, PCL10 and FUR4 promoters are bound by Gal4 when cells are grown in galactose but not in glucose ( Figure 6A).
  • the binding of Gal4p to the MTH, PCL10 and FUR4 promoters was verified by conventional ChIP analysis ( Figure 6C).
  • MTHl and MTH, PCL10 and FUR4 as Gal4-regulated genes reveals how regulation of several different metabolic pathways are interconnected (Figure 6F).
  • MTHl encodes a transcriptional repressor of many genes involved in metabolic pathways that would be unnecessary when cells utilize galactose as a sole carbon source.
  • HTX hexose transport
  • the results described herein indicate that the cell responds to galactose by modifying (increasing) the concentration of its galactose transporters at the membrane in a Gal4-dependent fashion at the expense of other transporters,
  • Gal 4 activates expression ofthe galactose transporter gene GAL2
  • Gal4 induction ofthe MTHl repressor gene leads to reduced levels of glucose transporter expression.
  • the Pel 10 cyclin associates with Pho85p and appears to repress the formation of glycogen.
  • PCL10 is Gal4-activated indicates that reduced glycogenesis occurs to maximize the energy obtained from galactose metabolism.
  • FUR4 encodes a uracil pennease and its induction by Gal 4 may reflect a need to increase intracellular pools of uracil to permit efficient uridine 5'-diphosphate(UDP) addition to galactose catalyzed by Gal 7.
  • Gal 4 binds to at least some GAL gene promoters when cells are grown on carbon sources other than galactose, as long as glucose is absent. Genome-wide location analysis of Gal 4 in cells grown on raffinose was repeated and it was found that the results were essentially identical to those obtained when cells were grown on galactose. These results indicate that Gal 4 exhibits the same binding behavior at all its genomic binding sites and demonstrate that the genome-wide location method is highly reproducible.
  • Stel2 The genome-wide binding profile ofthe DNA-binding transcription activator Stel2 was also investigated.
  • Stel2 is of interest because it has a defined cellular role - it is key to the response of haploid yeast to mating pheromones - but only a few genes regulated by Stel 2 have been identified.
  • Activation ofthe pheromone- response pathway by mating pheremones causes cell cycle arrest and transcriptional activation of more than 200 genes in a Stel2-dependent fashion.
  • Stel2 is directly regulated by Stel 2 and which are regulated by /059371
  • FUS3 and STE12 encode components ofthe signal transduction pathway involved in the response to pheromone (Madhani et al, Trends Genet., 14:151 (1999)); AFR1 and GIC2 are required for the formation of mating projections (Konopka et al, Mol. Cell Biol, 13:6876 (1993); Brown et al, Genes Dev, 77/2972 (1997); Chen et al, Genes Dev., 77/2998 (1997)); FIG2, AGA1, FIG1 and FUS1 are involved in cell fusion (Erdman et al, J.
  • Stel2 binds to some promoters in the absence of pheromone signaling, however, its binding to most genes is enhanced by alpha factor.
  • Stel2p is bound to its own promoter both before and after pheromone treatment.
  • the binding and expression data argue that the regulation ofthe STE 12 gene involves a positive feedback loop. STE 12 expression is increased immediately after pheromone treatment, indicating that the bound but inactive Ste 12 activator is rapidly converted to an active form. Increased expression of STE 12 gene would allow more Stel2p to be made and this would, in turn, activate its genes.
  • the Pcl2-Pho85 and PCl l-Pho85 complexes act in concert with Clnl-Cdc28 and Cln-2-Cdc28 cyclin dependent kinase complexes to promote Glcell cycle progression (Measday et al, 1994).
  • the Pcl2-Pho85 kinase complex has a substrate specificity that is overlapping but different from that ofthe Clnl-Cdc28 and Cln2-Cdc28.
  • haploid yeast cells are arrested at start ofthe late Gl phase, due to the inhibition of Clnl-Cdc28 and Cln2-Cdc28 activities by Farl, which is encoded by another Ste 12 target gene.
  • Activation of PCL2 by Ste 12 after pheromone treatment indicates that increased Pho85 complex activities are likely necessary to compensate for the loss of Cdc28 activities.
  • Stel2 target genes identified by analysis of genome locations of Stel2 and expression profiles during pheromone induction encode proteins involved in various steps of the mating response. Among them are 11 previously uncharacterized. The cellular roles for these genes, including YNL279W, YOR129C, YOR343C, YPL192C, YER019W, YIL083C, YEL037C, YIL169C, YNL105W, YOL155C and YNR064C, are therefore most likely related to mating.
  • CSH1, PCL2, ERG24, SPC25, HYM1, and PGM1 encode proteins involved in cell wall biosynthesis, cell morphology, membrane biosynthesis, nuclear congression and regulation of gene expression. Furthermore, /059371
  • YER019W, YOR129C and SCH9 are among genes that are cell cycle regulated (Spellman et al, Mol. Cell Biol, P/3273 (1999).
  • the genes that are regulated by Ste 12 can be divided into two classes: those bound by Ste 12 both before and after pheromone exposure (e.g., STE 12, PLC2, FIG2 and FUS1), and those bound by Stel2 only after exposure to pheromone (e.g., CKI1 and CHS1).
  • the first class of genes is induced immediately after pheromone exposure, most likely by a mechanism that converts an inactive DNA-bound Ste 12 protein to an active transcriptional activator. This could take place by removal of repressors of Stel2 such as Digl/Rstl and Dig2/Rst2 (Olson et al, Mol. Cell Biol, 20:4199 (2000)).
  • induction of transcription is relatively slow. In this case, the binding of Ste 12 appears to be limited before pheromone exposure. It is also possible that the epitope tag on Ste 12 is masked at these promoters before pheromone treatment, perhaps due to the presence of additional regulatory proteins.
  • Stel2 has also been implicated in other cellular processes. Together with Tecl, Ste 12 regulates the filmamentation of diploid cells and invasive growth in haploids. Two genes, TEC1 and FLO11, have been identified as Stel2 targets in filamentous growth pathway. Ste 12 binding to these genes either in the presence or absence of alpha factor was not detected. It is likely that Stel2p's binding to these promoters is regulated by different physiological conditions.
  • a combination of genome-wide location and expression analysis can identify the global set of genes whose expression is controlled directly by transcriptional activators in vivo.
  • the application of location analysis to two yeast transcriptional activators revealed how multiple functional pathways are coordinately controlled in vivo during the response to specific changes in the extracellular environment. All ofthe known targets for these two activators were confirmed, and functional modules were discovered that are regulated directly by these factors.
  • Tr>W n Consensus Binding Motifs of Promoters Sound by Y ⁇ ast Cell C vcl * Transcriptional Regulators. y
  • the cell cycle activators Swi4, Mbpl, Swi5, Fkhl, Fkh2, Nddl Mcml, and Ace2 were tagged with a multicopy myc epitope by inserting the epitope coding sequence into the normal chromosomal loci of these genes.
  • Vectors developed by Cosma et al. Cell, 97:299-311 (1998) were used for amplifying a fragment that contains the repeated myc tag coding sequence flanked by 50 bp from both sides of the stop codon ofthe gene.
  • the PCR products were transformed into the 303 strain Z1258 (MAT ⁇ , ada2-l, lrpl-1, canl-100, leu2-3, 112, his3-l l, 15, ura3) to generate the tagged strains (Z1335, Z1372, Z1373, Z1446, Z1370, Z1369, Z1321, and Z1371, respectively).
  • Clones were selected for growth on TRP plates, the insertion ofthe tagged sequence was confirmed by PCR, and expression ofthe epitope-tagged protein was confirmed by Western blotting using an anti-Myc antibody (9E11).
  • a strain containing a myc-tagged version of Swi5 was obtained from K. Nasmyth).
  • the enriched DNA was amplified and labeled with a fluorescent dye (Cy5) with the use of a ligation-mediated polymerase chain reaction (LM-PCR).
  • Cy5 ligation-mediated polymerase chain reaction
  • Cy3 ligation-mediated polymerase chain reaction
  • IP immunoprecipitation
  • Cy3 and Cy5 fluorescence intensities were generated by scanning the arrays using a GSI Lumonics Scanner.
  • the Cy3 and Cy5 images were analyzed using ArrayVision software, which defined the grid of spots and quantified the average intensity of each spot and the surrounding background intensity. The background intensity was subtracted from the spot intensity to give the final calculated spot intensity. The intensity ofthe two channels was normalized according to the median. For each spot, the ratio of corrected Cy5/Cy3 intensity was computed.
  • the intergenic regions present on the array were assigned to the gene or genes found transcriptionally downstream. Where a single intergenic region contains promoters for two divergently transcribed genes, the intergenic region was assigned to the gene or genes expressed during the cell cycle according to the Spellman et al. Mol. Cell Biol. Cell, P/3273-3297 (1998) analysis. The Spellman et al 1998 analysis was chosen because it incorporates all available yeast cell cycle expression data. Promoter regions detected with a p value O.001 were included for further analysis.
  • Genome-wide location analysis (Ren et al, Science, 290:2306-2309 (2000)) was used to identify the in vivo genome binding sites for each ofthe known cell cycle transcription factors ( Figures 9A and 9B).
  • Yeast strains each containing a myc-tagged version of Mbpl, Swi4, Swi6, Mcml, Fkhl, Fkh2, Nddl, Swi5, or Ace2, were grown in asynchronous cultures to mid log phase and subjected to location analysis as described previously (Ren et al, Science, 290:2306-2309 (2000)).
  • Mbpl, Swi4, and Swi6 bound predominantly to promoter regions of late Gl genes (p ⁇ 10 '14 , p ⁇ 10 "18 , and p ⁇ 10 "20 respectively), Swi5 and Ace2 to M Gl genes (p ⁇ 10 "14 and p ⁇ 10 "3 , respectively), and Mcml, Fkh2, and Nddl to G2/M genes (p ⁇ 10 "14 , p ⁇ 10 '15 , and p ⁇ 10- 21 , respectively).
  • the data described herein generally support the model for stage-specific regulation of gene expression by these activators and extend it to encompass promoters for several hundred cell cycle genes.
  • SWI4 is regulated by Mcml and Swi6 (Foster et al, Mol. Cell Biol, 75/3792-3801 (1993); Mackay et al, Mol. Cell Biol, 27/4140-4148 (2001); Mclnerny et al, Genes Dev., 77/1277-1288 (1997)), Swi5 is regulated by Mcml/Fkh2/Nddl complex (Koranda et al, Nature, 406:94-98 (2000); Kumar et al Curr.
  • the regulatory network from the genomic binding data ( Figure 1 IB) described herein can be described as follows.
  • SBF Spi4/Swi6
  • MBF Mbpl/Swi6
  • Mcml and Fkh2 are bound to promoters throughout the cell cycle, and activation of G2/M genes is dependent on recruitment of Nddl (Koranda et al. Nature, 406:94-98 (200O)).
  • the Mcml/Fkh2/Nddl complex regulates SWI5 and ACEL Swi5, Ace2, and Mcml activate /G7 genes.
  • Mcml binds to the SWL4 promoter and contributes to its activation in M/Gl, leading to accumulation ofthe Swi4 subunit ofthe SBF transcription factor in Gl .
  • All three M/Gl transcription factors regulate CLN3, whose protein product forms a complex with Cdc28, which in turn activates SBF and MBF during late Gl (Dirick et al. Embo. J., 74/4803-4813 (1995)).
  • Swi4 transcription is further regulated in late Gl by both SBF and MBF.
  • the serial regulation of cell cycle regulators occurs throughout the cycle, forming a fully connected regulatory network that is itself a cycle.
  • SBF and MBF control transcription of Gl/M cyclin genes, but also regulate expression ofthe G2/M cyclin Clb2, which inhibits further expression ofthe Gl/S cyclins Cinl and Cin2 (Amon et al. Cell, 74/993-1007 (1993)) and promotes entry into mitosis (Surana et al. Cell, (55/145-161 (1991)).
  • SBF and MBF also regulate the transcription ofthe transcription factor Nddl, which also binds the CLB2 promoter.
  • Nddl which also binds the CLB2 promoter.
  • SBF, MBF and Nddl ultimately collaborate to regulate transcription ofthe CLB2 gene.
  • SBF and MBF therefore regulate genes necessary for the transition through Gl/S, as well as genes whose products set the stage for further progression through the cell cycle.
  • the data also reveal that the G2/M activators (Mcml/Fkh2/Nddl) bind genes whose expression is necessary for both entry into and exit from mitosis.
  • the G2/M activators bind and regulate transcription of CLB2, whose product is necessary to enter mitosis (Surana et al. Cell, (55/145-161 (1991)). They also set the stage for exit from mitosis by regulating the gene encoding Cdc20, an activator ofthe APC, which targets the APC to degrade Pdsl and thus initiate chromosome separation (Nisintin et al. Science, 278:450-463 (1997)).
  • Cdc20-activated APC also degrades Clb5 (Shirayama et al. Nature, 402:203-207 (1999)) and thus enables Cdcl4 to promote the transcription and activation of Sicl (Shirayama et al. Nature, 402:203-207 (1999)) and to initiate the degradation of Clb2 (Jaspersen et al, Mol. Biol. Cell, 0:2803-2817 (1998); Nisintin et al, Science, 275/450-463 (1997)).
  • the G2/M activators Mcml/Fkh2/ ⁇ ddl regulate transcription of SPO12, which encodes a protein that also regulates mitotic exit (Grether et al, Mol. Biol. Cell, 10:3689- 2703 (1999)).
  • the M/Gl transcriptional regulators (Mcml, Ace2, and Swi5) bind genes that are key to entering and progressing through Gl .
  • Swi5 binds to the S7C7 promoter, and all three transcriptional regulators bind to the GLN3 promoter.
  • Sicl inhibits Clb-Cdc28 during mitosis (Toyn et al. Genetics, 145:85-96 (1997)), thus facilitating exit from mitosis.
  • Cln3-Cdc28 activates SBF and MBF in late Gl (Dirick et al. Embo. J, 74:4803-4813 (1995)), thus setting the stage for another cell cycle circuit.
  • knowledge ofthe global set of cyclin and CDK regulatory genes that are bound by each ofthe transcriptional activators provides a much enriched model to explain how transcriptional regulation contributes to cell cycle progression (Figure 12B).
  • SBF regulates genes involved in the morphological changes associated with cell budding
  • MBF controls genes involved in DNA replication and repair, confirming a previous study (Iyer et al, Nature, 409:533-536 (2001)).
  • SBF is also bound to the promoters of several histone genes (HTA1, HTA2, HTA3, HTB1, HTB2 and HHO1), which makes it likely that SBF contributes to the increase in histone gene transcription observed at S phase.
  • Fkhl was found to bind various genes that encode proteins associated with chromatin structure and its regulation; these include histones (HHFl and HHT1), telomere length regulators (TEL2 and CTF18), a shared component of the chromatin remodeling complexes Swi/Snf and RSC (ARP7), and a histone deacetylase (HOS3).
  • the G2/M activators (Mcml/Fkh2/Nddl) bind genes that regulate the transition through mitosis (SWI5, ACE2, CLB1, CDC20 and SP012).
  • EGT2 + + Cell-cycle regulation protein may be involved in cytokinesis
  • STE2 + Pheromone alpha-factor receptor has seven transmembrane segments
  • a full list of target genes is available at the author's web site (http //web wi mit edu/young/cellcycle)
  • the DNA replication category includes genes that function in DNA synthesis, in DNA repair and in sister chromatid cohesion
  • the factor location data demonstrate that each ofthe nine cell cycle transcription factors binds to critical cell cycle genes, yet cells with a single deletion of MBP1, SWI4, SWI6, FKH1, FKH2, ACE2, or SWI5 are viable; only MCM1 and NDD1 are essential for yeast cell survival (Breeden Curr. Biol, 70.-R586- R588(2000); Loy et ⁇ l Mol. Cell Biol, 7P.-3312-3327 (1999); Mendenhall et ⁇ l, Mol. Biol. Rev., (52:1191-1243 (1998)).
  • the conventional explanation for this observation is that each nonessential gene product shares its function with another.
  • Swi4 and Mbpl share 50% identity in their DNA binding domains (Koch et ⁇ l Science, 261:1551-1557 (1993)). Similarly, Fkhl and Fkh2 are 72% identical (Kumar et ⁇ l. Curr. Biol, 70:896-906 (2000)), and Swi5 and Ace2 are 83% identical in their respective DNA binding domains (McBride et ⁇ l. J. Biol Chem., 274:21029- 21036 (1999)). Each of these pairs of proteins recognizes similar DNA motifs, so it is likely that functional redundancy rescues cells with mutations in individual factors. However, it was not clear whether each ofthe pairs of factors had truly redundant functions in normal cells, or whether they exhibit redundant function only in mutant cells that lack the other factor.
  • a key concept that emerged from this study is that cell cycle transcriptional control is effected by a connected regulatory network of transcriptional activators.
  • the cell cycle transcriptional regulators that function during one stage ofthe cell cycle regulate the transcriptional regulators that function during the next stage, and this serial regulation of transcriptional regulators forms a complete regulatory circuit.
  • the transcriptional regulatory network that controls the cell cycle is itself a cycle of regulators regulating regulators.
  • the discovery of this connected transcriptional regulatory network is important for several reasons. It provides additional understanding ofthe regulatory mechanism by which cells ensure transitions from one stage into the appropriate next stage. It supplies the foundation for future work on the mechanisms that coordinate gene expression and other aspects of cell cycle regulation. Furthermore, it suggest that a connected, circular transcriptional regulatory network is likely a fundamental feature of cell cycle regulation in other, more complex, organisms.
  • the results described herein identify how the cyclin genes regulated, by the nine transcriptional activators.
  • the results reveal that transcription factors that regulate the cyclin genes during each phase ofthe cell cycle also regulate genes that are involved in transitioning to the next stage ofthe cycle ( Figures 12A- 12B).
  • the Gl/S activators SBF and MBF control transcription of Gl/S cyclin genes, but also regulate expression of G2/M cyclin Clb2, which subsequently inhibits further expression ofthe Gl/S cyclins Cinl and Cin2 and promotes entry into mitosis.
  • the cell cycle transcriptional regulatory network has evolved so that some transcriptional regulators contribute to the control of both stage entry and exit.
  • the identification of sets of genes that are bound by each of these regulators reveals how coordinate regulation of a wide variety of stage-specific cell cycle functions is regulated ( Figure 13).
  • the Gl/S activators regulate genes involved in cell budding, DNA replication and repair, and chromosome maintenance.
  • the G2/M activators bind genes that regulate transition through mitosis.
  • the late M factors regulate genes involved in cytokinesis and prereplication complex formation.
  • the Swi4 regulator becomes functionally active at START, via a mechanism that is dependent on Cln3-Cdc28, when the cell reaches a critical size (Dirick et al, Embo. J., 74:4803-4813 (1995)).
  • the SWL4 promoter is bound by Swi4 itself, indicating that a positive feedback loop exists to ensure that adequate levels of Swi4, and thus, SBF, are present prior to commitment.
  • the observation that the Gl/S regulators SBF and MBF both regulate NDD1 suggests how adequate levels of Nddl are produced to initiate the G2/M transcriptional program.
  • Nddl protein is a limiting component ofthe complex that activates G2/M genes; Mcml and Fkh2 are bound to promoters throughout the cell cycle, and activation of G2/M genes is dependent on recruitment of Nddl (Koranda et al, Nature, 406:94-98 (2000).
  • the Mcml/Fkh2/Nddl complex regulates SWL5 and ACE2, whose products become functional only in late anaphase after relocalization to the nucleus in a mechanism that is dependent on low Clb-Cdc28 activity (Nasmyth et al, Cell, 62:631-647 (1990); Shirayama et al, Nature, 402:203-207 (1999)). Later in the cell cycle, the Swi5, Ace2, and Mcml factors all bind to the CLN3 promoter, thus assuring adequate levels ofthe Cln3 cyclin at START.
  • the cell cycle transcriptional regulatory network model accounts for several observations relevant to cell cycle regulation.
  • the use of multiple transcription factors to regulate key transcription and cyclin regulators explains why mutations in single transcription factors generally have only limited effects on progression through the cell cycle, whereas mutations in activator pairs can have substantial effects (Breedon, Curr. Biol, 70:R586-R588(2000); Koch, et al, Science, 261:1551-1557 (1993); Mendenhall et al, Mol. Biol. Rev., (52:1191-1243 (1998)).
  • Nutrient limitation causes yeast cells to arrest cell cycle progression, but rather than counting at the time of nutrient limitation, the arrest is delayed until the cells reach Gl (Mendenhall et al, Mol.
  • the location data presented herein are well adapted to new computational approaches to discovering genetic regulatory networks.
  • the binding of a transcriptional activator to the promoter region of a gene indicates that the activator has a regulatory effect on the gene.
  • location information must be fused with other data, such as expression data, to fully elaborate the complete mechanism of transcriptional regulation and the form of regulatory networks.
  • New computational approaches will synergistically combine location data with other data types to form a well-focused picture of cellular function. For example, one way to combine location and expression data is to use the location data to first suggest tentative factor-target pairs with associated p-values.
  • the cell cycle activators Swi4, Mbpl, Swi6, Fkhl, Fkh2, Nddl, Mcml and Ace2 were tagged with a 9 or 18 copy myc epitope by inserting its coding sequence into the normal chromosomal loci of these genes.
  • Vectors developed by K Nasmyth was used for recombination ofthe epitope coding sequence into the W303 strain Z1256.
  • the specific oligonucleotides used to generate PCR products are described here.
  • the PCR products were transformed into the strain Z1256 to generate the tagged strains.
  • Clones were selected for growth on TRP- plates, and the insertion was confirmed by PCR and expression ofthe epitope-tagged protein was confirmed by western blotting using an anti-Myc antibody (9E11).
  • a 9 myc tagged version of Swi5 (Z1407) was obtained from K Nasmyth.
  • the chromatin imunoprecipitation part of that protocol is based on a protocol obtained from the Nasmyth lab and one from Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. , "Spreading of transcriptional repressor SIR3 from telomeric heterochromatin,” Nature 383,92-6 (1996).
  • the Nasmyth protocol was optimized for use with W303 ⁇ strains tagged with a Myc 18 epitope inserted at the C-terminus of various transcription factors (strains obtained from Pia Cosma).
  • MATa, ade2-l, t ⁇ l-1, canl-100, leu2-3,l 12, his3-l 1,15, ura3, GAL+,psi+, Z1373
  • MATa, ade2-l, trpl-1, canl-100, leu2-3,112, his3-l l,15, ura3, GAL+,psi+, Z1448
  • MCM1 18-Myc-MCM1
  • ACE2 18-Myc-ACE2
  • the genome-wide location analysis method allows protein-DNA interactions to be monitored across the entire yeast genome.
  • the method combines a modified Chromatin Immunoprecipitation (ChIP) procedure, which has been previously used to study in vivo protein-DNA interactions at one or a small number of specific DNA sites (Aparicio, O.M., in Current Protocols in Molecular Biology. F. M. Ausubel, et al., Eds. (John Wiley and Sons, Inc., New York, 1999) pp.
  • ChIP Chromatin Immunoprecipitation
  • a sample of DNA that has not been enriched by immunoprecipitation is subjected to LM-PCR in the presence of a different fluorophore, and both IP-enriched and unenriched pools of labeled-DNA are hybridized to a single DNA microarray containing all yeast intergenic sequences.
  • the IP-enriched/unenriched ratio of fluorescence intensity obtained from three independent experiments and a p-value is assign to each spot according to an error model adapted from Roberts, CJ., et al, "Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles," Science 287,873-80 (2000).
  • the average ratio is then calculated using a weighted average analysis method, providing the relative binding ofthe protein of interest to each sequence represented on the array.
  • Yeast Intergenic DNA Array Using the Yeast Intergenic Region Primer set (Research Genetics) we PCR amplified and printed 6361 spots, representing essentially all ofthe known intergenic regions in the yeast genome. The average size ofthe spotted PCR products was 480 bp, and the sizes ranged from 60 bp to 1500 bp.
  • Yeast cells expressing an epitope-tagged protein of interest were used; a Myc- epitope coding sequence was integrated into the genome at the 3'-end ofthe coding sequence for each protein.
  • Cultures of yeast cells were grown to OD600 of 0.8 under appropriate conditions prior to formaldehyde crosslinking.
  • DNA amplification and labeling with LM-PCR was found to produce more reproducible results relative to amplification of enriched DNA as a library in E. coli. Superior and more reproducible results were also obtained when DNA preparations enriched by ChIP were compared to unenriched DNA preparations (rather than DNA preparations obtained from an untagged strain subjected to ChLP).
  • the 6361 intergenic regions were amplified using the Yeast Intergenic Region Primers (Research Genetics) primer set. 50 ⁇ L PCR reactions were performed in 96- well plates with each primer pair with the following conditions: 0.25 ⁇ M of each primer, 20 ng of yeast genomic DNA, 250 ⁇ M of each dNTP, 2 mM MgC12, IX PCR buffer (Perkin Elmer), and 0.875 units of Taq DNA polymerase (Perkin Elmer). PCR amplification was performed in MJ Research Thermocyclers beginning with 2 minute denaturation at 95°C, followed by 36 cycles of 30 seconds at 92°C, 45 seconds at 52°C, and 2 minutes at 72°C, with a final extension cycle of 7 minutes at 72°C.
  • the resuspended DNA was transfered to 384 well plates and printed on GAPS-coated slides (Corning) using a Cartesian robot (Cartesian Technologies).
  • the printed slides were rehydrated, snap-dried, and UN crosslinked in UV Stratalinker (Stratagene) set at 60 mJoules.
  • the slides were then stored under vacuum for at least 2 days prior to hybridization.
  • Step 1 Preparation of cells and cross linking
  • Step lb Washing and storage of cells
  • Vibrax-VXR at maximum power for 2 hours at 4°C.
  • Step 3a Elution from beads and reversal of cross links
  • Extract 2 times with 1 volume of phenol (Sigma Cat. P-4557; OK to use at 4°C). Spin for about 5 minutes at room temperature for each extraction.
  • step 5 Store at -20°C or place on ice and proceed to step 5.
  • Step 5 Blunting DNA
  • the program name is "12/20", under "Main” in the 2 heads PCR machine. Do not use the heated lid option.
  • Step 5 a - Ligation of blunt DNA to linker Add 25 ⁇ l of cold ligase mix:
  • PCR product should be a smear ranging from 200 bp to 600 bp with an average size of 400 bp).
  • Pipette 50 ⁇ l of probe onto slide and drop cover slip (use the big one so that it will cover the entire array) onto the liquid. Try to avoid bubbles as they exclude the hybridization solution.
  • the 9E11 antibody we are using has been purified from acites and concentrated. The amount used has been determined empirically so that the beads are saturated. Spin for 1 minute at speed 6 (-3000 ⁇ m) in a tabletop centrifuge (Sorvall RT6000).
  • oJW102 GCGGTGACCCGGGAGATCTGAATTC (SEQ ID NO: 29)
  • oJW103 GAATTCAGATC (SEQ ID NO: 30)
  • Lysis Buffer make fresh with cold ddH 2 0
  • Pepstatin mix 100X (aliquot and store at -20°C)
  • DNA microarrays with consistent spot quality and even signal background were important for maximizing reproducibility and dynamic range.
  • Cy3 and Cy5 fluorescence intensities were generated by scanning the arrays using a GSI Lumonics Scaimer.
  • the Cy3 and Cy5 images were analyzed using ArrayVision software, which defined the grid of spots and quantified the average intensity of each spot and the surrounding background intensity. The background intensity was subtracted from the spot intensity to give the final calculated spot intensity.
  • the intensities of all ofthe spots from the Cy5 and Cy3 scans were summed, and the ratio of total Cy5/Cy3 intensity was set equal to one. For each spot the ratio of corrected Cy5/Cy3 intensity was computed.
  • a, are the intensities measured in the two channels for each spot
  • ⁇ 1>2 are the uncertainties due to background subtraction
  • f is a fractional multiplicative error such as would come from hybridization non-uniformities, fluctuations in the dye inco ⁇ oration efficiency, scanner gain fluctuations, etc.
  • X is approximately normal.
  • the parameters ⁇ and f were chosen such that X has unit variance. The significance of a change of magnitude
  • is then calculated as
  • the method to combine repeated measurements of chromosomal binding is adapted, with a few modifications, from a method by developed by Roberts, C.J., et al., " Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles," Science 287,873-80 (2000) to average multiple measurements of gene expression.
  • the binding ratio is expressed as the log 10 (a 2 /a 1 ), where a,,a 2 are the intensities measured in the two channels for each spot.
  • ⁇ is the error of log 10 (a 2 /a,) from (3), x f stands for i-th measurement of log 10 (a 2 /a,), n is the number of repeats.
  • the error of x can be computed in two ways. One is to propagate the errors ⁇ , , another is from the scatter of x,:
  • the intergenic regions present on the array were assigned to the gene or genes found transcriptionally downstream.
  • a single intergenic region contains the promoter for two divergently transcribed genes (e.g. HHF2 and HHT2 or CLN2 and BBP1).
  • the intergenic region was assigned to both genes, and gene expression data were used to "discipline" the binding location data. This was accomplished by selecting genes whose promoters were bound by factors and whose expression oscillates during the cell cycle. Among genes whose promoters were bound by at least one ofthe factors and which were expressed in a cell cycle- dependent fashion, we found only 18 examples of intergenic regions that lie at the center of divergently transcribed genes.
  • the raw data for the location analysis experiments for each ofthe nine cell cycle activators are available as a single text file with each column separated by tabs. Descriptions ofthe contents of each column are provided in the first two rows.
  • 'spot name' refers to an intergenic region. It has been assigned a systematic name that includes the letter 'i' followed by the systematic ORF name that is to the left of the intergenic region.
  • 'per quality' is a qualitative description ofthe per products as seen on an acrylamide gel. 'good' means that the band was the correct size and cleary visible, 'w' indicates that the band intensity was 'weak', 'vw' indicates 'very weak' intensity, 'no' means that no band was seen, and 's' indicates that the size ofthe band was not what was expected.
  • '# of promoters on spot' denotes the number of genes which the intergenic region contains promoters for.
  • 'assigned gene' is the name of each orf whose promoter is contained in the given intergenic region, 'Orf is the gene name.
  • 'p-value' and 'average ratio' are the combined values for replicate experiments for each ofthe factors tested.
  • the last columns in the file are the cell cycle stage as described by Spellman, P. T., et al, Mol Biol Cell 9, 3273-97 (1998), the phase ofthe gene, and the cell cycle stage as described by Cho, R. J., et al, Mol Cell 2, 65-73 (1998).
  • Genomic binding sites were identified for the nine known yeast cell cycle transcription activators, revealing how these factors coordinately regulate global gene expression and diverse stage-specific functions to produce a continuous cycle of events.
  • One fundamental insight that emerged from these results is that a complete transcriptional regulatory circuit is formed by activator complexes that control next-stage activators.
  • the results also show that stage-specific activator complexes regulate genes encoding CDK regulators necessary for both stage entry and for progression into the next stage ofthe cell cycle. This global information provides a map ofthe regulatory network that controls the cell cycle.
  • In vivo binding which includes chromatin immuno-precipitation and in vivo footprinting.
  • Genetic analysis which includes the effects of genetic manipulations (such as mutations or overproduction) on target genes.
  • Sequence analysis which includes the identification of DNA binding motifs in the promoters of target genes.
  • Swi6 as a cofactor for Swi4 (SBF) and Mbpl (MBF)
  • Swi6 has been ' shown to function as a subunit of both SBF and MBF (Dirick et al. Nature, 357/508-513 (1992)).
  • the genome-wide location analysis data indicates that Swi6 binds to almost all ofthe promoter regions bound by Mbpl and Swi4 ( Figure 2A), indicating that it is a co-factor of these two regulators throughout the genome.
  • SBF and MBF included key cell cycle regulators (Table 5). SBF and MBF were found to bind the promoters of CLN1, CLB6 and PCL1, SBF binds the promoters of CLN2 and PCL2 and MBF binds the promoter of CLB6.
  • the location analysis also shows that SBF participates in the regulation of G2/M cyclin (Clb2) activity at three levels. First, as suggested previously (Iyer et al. Nature, 409:533- 536 (2001)) it binds and presumably directly regulates CLB2. Second, SBF regulates the transcription ofthe transcription factor Nddl, which in rum also regulates CLB2 transcription.
  • SBF and Nddl collaborate to regulate transcription of the CLB2 gene, whose product is necessary to enter mitosis.
  • SBF and MBF regulate SWE1 and GIN4.
  • Swel is an inhibitor of Cdc28-Clb2 which delays entry into mitosis in response to bud emergence defects (Sia, R.
  • SBF and MBF participate in the regulation of genes essential for cellular functions specific to late Gl.
  • SBF regulates genes involved in the morphological changes associated with cell budding and MBF controls genes involved in DNA replication and repair (Table 5), confirming a previous study (Iyer et al. Nature, 409:533-536 (2001)).
  • HTA1, HTA2, HTA3, HTB1, HTB2 and HHOi histone genes
  • SWI4 nor MBP1 is essential for cell viability, but a SWI4 IMBP1 double mutant is lethal, suggesting that some redundancy exists between Swi4 and Mbpl (Mendenhall, M. D., and ⁇ odge, A. E., "Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle ofthe yeast Saccharomyces cerevisiae," Microbiol Mol Biol Rev 62, 1191-243 (1998)).
  • SBF single-dependent protein kinase activity during the cell cycle ofthe yeast Saccharomyces cerevisiae
  • Promoter binding motifs The large number of targets we found enabled us to search for putative DNA binding motifs. To this end we ran AlignACE (Hughes, J. D., et al, JMol Biol 296, 1205-14 (2000)), a program that uses a Gibbs sampling algorithm to find common regulatory elements among a collection of promoters. We found a refined version of the known binding sites of Swi4 and of Mbpl . Although these motifs are highly enriched in the set of target genes identified by our location analysis (p ⁇ 0- ⁇ 4 and p ⁇ 0-20 respectively), they also occur in the promoters of many genes that show no evidence of binding to these factors in vivo, suggesting that the presence of this sequence alone is not a predictor of factor binding.
  • Fkhl and Fkh2 are two members of the Forkhead family of proteins that share 82% similarity in amino acid sequence (Kumar et al. Curr. Biol, 70:896-906 (2000)). Genetic analysis has suggested that these two genes are involved in cell cycle control, in pseudohyphal growth, and in silencing of HMRa (Hollenhorst et al. Genetics, 154:1533- 1548 (2000)). Their contribution to the regulation of cell cycle genes appears to be in G2/M, since it has been shown that Fkh2, together with Mcml, recruits Nddl and thereby regulates the G2/M specific transcription of CLB2, SWI5 and YJL051 W (Zhu et al.
  • Fkhl and Fkh2 are involved in regulating genes expressed in G2/M, but indicate that these proteins also regulate genes expressed in other cell cycle stages.
  • Fkh2 binds predominantly to promoters of genes expressed in G2/M (p ⁇ 0-9), but it is also enriched in Gl (p ⁇ 0-4) and S/G2 (p ⁇ 10-3).
  • Fkhl target genes are expressed in Gl (p ⁇ 0-2), S (p ⁇ 0-3), S/G2 (p ⁇ 0-5) and G2/M (p ⁇ 0-4).
  • the association of Fkhl or Fkh2 with Mcml is limited to genes expressed in G2/M; in other stages Fkhl and Fkh2 bind to promoters in the absence of Mcml.
  • Fkhl and Fkh2 include several key cell cycle regulators (Table 5).
  • Fkh2 bound to the promoter of HSL7 which encodes a regulator of Swel that is necessary for the transition into mitosis (Shulewitz, M. J., et al, Mol Cell Biol 19, 7123-37 (1999)).
  • Fkhl and Fkh2 also bind to promoters of genes involved in exit from mitosis; these include APC1, which encodes for a component ofthe anaphase-promoting complex (Zachariae, W., and Nasmyth, K. Genes Dev 13, 2039-58 (1999)), and TEM1, which encodes a protein required for activation of Cdcl4p and the mitotic exit pathway (Krishnan, R., et al. Genetics 156, 489-500 (2000)).
  • APC1 which encodes for a component ofthe anaphase-promoting complex
  • TEM1 which encodes a protein required for activation of Cdcl4p and the mitotic exit pathway
  • Fkhl was found to bind various genes that encode proteins associated with chromatin structure and its regulation; these include histones (HHFl and HHT1), telomere length regulators (TEL2 and CTF18), a component ofthe chromatin remodeling complexes Swi/Snf and RSC (ARP7), and histone deacetylase (HOS3).
  • HHFl and HHT1 histones
  • TEL2 and CTF18 telomere length regulators
  • ARP7 histone deacetylase
  • Fkhl and Fkh2 have distinct roles in cell cycle progression, but redundant roles in pseudohyphal growth (Hollenhorst et al. Genetics, 154:1533-1548 (2000)).
  • Fkhl and Fkh2 bind to the promoters of 38 and 56 cell cycle genes, respectively, and that 16 of these genes were bound by both proteins.
  • G2/M genes that are targets of Fkh2
  • three genes (CLB2, ACE2 and BUD4) are also targets of Fkhl.
  • Promoter binding motifs In order to identify the binding motifs for Fkhl and Fkh2, we ran AlignACE (Hughes, J. D., et al, JMol Biol 296, 1205-14 (2000)) on the set of promoters bound by each factor.
  • the program identified the known Forkhead binding motif (GTAAACAA (SEQ ID NO: 31)) in the two sets of promoters (p ⁇ 10-9). However, this sequence was absent from most ofthe promoters bound by Fkhl and Fkh2, suggesting that additional sequence elements contribute to the binding sites for these proteins.
  • Mcml is involved in the regulation of cell cycle genes that are expressed both in G2/M and in M/Gl. Mcml collaborates with Nddl and Fkhl or Fkh2 to regulate G2/M genes (Zhu et al. Nature, 406:90-94 (2000); Koranda et al, Nature, 406:94-98 (2000); Kumar et al. Curr. Biol, 10:896- 906 (2000); Pic et al. Embo , 79:3750-3761 (2000)). Mcml also regulates M/Gl genes, but less is known about its functions in this stage of the cell cycle (Mclnemy et al. Genes Dev., 77:1277-1288 (1997)).
  • Mcml target genes in G2/M and M/Gl are governed by Mcml 's association with different regulatory partners.
  • Mcml binds predominantly to promoters of genes in G2/M (p ⁇ 0- ⁇ 4) and in M/Gl (p ⁇ 0-6).
  • Mcml 's cofactors Nddl and Fkh2 bind to promoters of G2/M genes (p ⁇ 0-2 ⁇ and j? ⁇ 10-15 respectively) but were absent from promoters of M/Gl genes.
  • the G2/M activators regulate transcription of CLB2, whose product is necessary to enter mitosis. They also set the stage for exit from mitosis at several levels. First, they regulate the transcription of SWI5 and ACE2, which encode key M/Gl transcriptional activators. Second they bind the promoter of CDC20, an activator of the anaphase promoting complex (APC), which targets the APC to degrade Pdsl and thus initiate chromosome separation (Nisintin et al. Science, 7 ⁇ °:450-463 (1997)).
  • APC anaphase promoting complex
  • Cdc20-activated APC also participates in the degradation of Clb5 (Shirayama et al. Nature, 402:203-207 (1999)), and thus enables Cdcl4 to promote the transcription and activation of Sicl (Shirayama et al. Nature, 402:203-207 (1999)) and to initiate degradation of Clb2 (Jaspersen et al, Mol. Biol. Cell, 9:2803- 2817 (1998); Visintin et al, (1998)). Finally these activators regulate transcription of SPOl 2, which encodes a protein that also functions to regulate mitotic exit (Grether et al, Mol. Biol Cell, 10:3689-2703 (1999)).
  • Mcml in the absence of ⁇ ddl and Fkh2 binds the promoters of SWI4, a late Gl transcription factor, CLN3, a Gl cyclin that is necessary for the activation of Gl transcription machinery (Dirick et al. Embo. J., 74:4803-4813 (1995)) and FAR1, which encodes an inhibitor ofthe Gl cyclins (Valdivieso, M. H., et al. Mol Cell Biol 13, 1013-22 (1993)).
  • Mcml in the absence of ⁇ ddl and Fkh2, participates in the regulation of genes essential for cellular functions specific to late mitosis and early Gl . It binds to and apparently regulates genes encoding proteins involved in pre-replication complex formation (MCM3, MCM6, CDC6 and CDC46) and in mating (S7E2, STE6, FAR1, MFA1, MFA2, AGA1 and AGA2).
  • Ace2 and Swi5 have been shown to control certain genes expressed in late mitosis and early Gl phases ofthe cell cycle (McBride et al J. Biol. Chem., 274:21029- 21036 (1999)). Our results confirm that Ace2 and Swi5 bound predominantly to promoters of M/Gl genes (p ⁇ 0-3 and/ lO-14, respectively).
  • Ace2 and Swi5 included cell cycle regulators (Table 5), Ace2 bound to the promoter of PCL9, whose product is the only cyclin known to act in M/Gl (Aerne, B. L., Mol Biol Cell 9, 945-56 (1998)). Both Ace2 and Swi5 bound to promoters of two ofthe Gl cyclin genes (PCL2 and CLN3), and Swi5 bound to the gene encoding the cyclin regulator Sicl, which inhibits Clb-CDK activity, allowing exit from mitosis.
  • PCL2 and CLN3 two ofthe Gl cyclin genes
  • Ace2 and Swi5 were bound to the promoters of several genes whose products are involved in cell wall biogenesis and cytokinesis (Table 5). Swi5 bound to the promoters of 17 Y' genes, which are a subgroup of a larger group of sub-telomeric genes that share DNA sequence similarity and whose expression peaks in early Gl (Spellman, P. T., et al, Mol Biol Cell 9, 3273-97 (1998)). Redundancy of activators
  • Genome-wide location analysis identifies the set of promoters that are bound by the same transcription factor.
  • the availability of a large number of putative targets is ideal for DNA binding motif searching to identify common DNA regulatory elements.
  • AlignACE program In order to identify the consensus binding sites for cell cycle transcription factors, we used the AlignACE program (Hughes, J. D., et al, J Mol Biol 296, 1205-14 (2000)).
  • Each activator group regulates at least one activator for the next phase. • Each activator group regulates genes involved in phase entry and CDK cyclin regulators that set the stage for exiting that phase.
  • partial redundancy between pairs of activators may serve to ensure that the cell cycle is completed efficiently while allowing each activator to regulate distinct functional groups of genes.

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Abstract

La présente invention porte sur un procédé d'identification d'une région (ou de plusieurs régions) d'un génome d'une cellule à laquelle (auxquelles) est liée une protéine. Selon les procédés décrits, la protéine de liaison à l'ADN d'une cellule est liée (c'est-à-dire réticulée de manière covalente) à l'ADN génomique d'une cellule. L'ADN génomique auquel la protéine de liaison est liée est retiré et combiné, ou mis en contact avec un ADN comprenant une séquence complémentaire à l'ADN génomique de la cellule dans des conditions où se produit l'hybridation entre l'ADN génomique identifié et la séquence complémentaire à l'ADN génomique. La ou les régions d'hybridation constituent la ou les régions du génome de la cellule où se lie la protéine. L'invention concerne également un procédé d'identification d'un ensemble de gènes où la liaison régulatrice de cycle cellulaire est en corrélation avec l'expression génique, ainsi qu'un procédé d'identification de cibles génomiques d'activateurs transcriptionnels de cycle cellulaire de cellules vivantes.
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KORANDA M. ET AL.: "Forkhead-like transcription factors recruit Ndd1 to the chromatin of G2/M-specific promoters", NATURE, vol. 406, 6 July 2000 (2000-07-06), pages 94 - 98, XP002225863, DOI: doi:10.1038/35017589 *
KUMAR R. ET AL.: "Forkhead transcription factors, Fkh1p and Fkh2p, collaborate with Mcm1p to control transcription required for M-phase", CURRENT BIOLOGY, vol. 10, no. 15, 12 July 2000 (2000-07-12), pages 896 - 906 *
See also references of WO02059371A3 *
SPELLMAN PT ET AL.: "Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization", MOLECULAR BIOLOGY OF THE CELL, vol. 9, December 1998 (1998-12-01), pages 3273 - 3297 *
ZHU G. ET AL.: "Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth", NATURE, vol. 406, 6 July 2000 (2000-07-06), pages 90 - 94 *

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