ChIP-on-chip

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Workflow overview of a ChIP-on-chip experiment. ChIP-on-chip workflow overview.svg
Workflow overview of a ChIP-on-chip experiment.

ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray ("chip"). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo . Specifically, it allows the identification of the cistrome, the sum of binding sites, for DNA-binding proteins on a genome-wide basis. [1] Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest. [1] As the name of the technique suggests, such proteins are generally those operating in the context of chromatin. The most prominent representatives of this class are transcription factors, replication-related proteins, like origin recognition complex protein (ORC), histones, their variants, and histone modifications.

Contents

The goal of ChIP-on-chip is to locate protein binding sites that may help identify functional elements in the genome. For example, in the case of a transcription factor as a protein of interest, one can determine its transcription factor binding sites throughout the genome. Other proteins allow the identification of promoter regions, enhancers, repressors and silencing elements, insulators, boundary elements, and sequences that control DNA replication. [2] If histones are subject of interest, it is believed that the distribution of modifications and their localizations may offer new insights into the mechanisms of regulation.

One of the long-term goals ChIP-on-chip was designed for is to establish a catalogue of (selected) organisms that lists all protein-DNA interactions under various physiological conditions. This knowledge would ultimately help in the understanding of the machinery behind gene regulation, cell proliferation, and disease progression. Hence, ChIP-on-chip offers both potential to complement our knowledge about the orchestration of the genome on the nucleotide level and information on higher levels of information and regulation as it is propagated by research on epigenetics.

Technological platforms

The technical platforms to conduct ChIP-on-chip experiments are DNA microarrays, or "chips". They can be classified and distinguished according to various characteristics:

Probe type: DNA arrays can comprise either mechanically spotted cDNAs or PCR-products, mechanically spotted oligonucleotides, or oligonucleotides that are synthesized in situ . The early versions of microarrays were designed to detect RNAs from expressed genomic regions (open reading frames aka ORFs). Although such arrays are perfectly suited to study gene expression profiles, they have limited importance in ChIP experiments since most "interesting" proteins with respect to this technique bind in intergenic regions. Nowadays, even custom-made arrays can be designed and fine-tuned to match the requirements of an experiment. Also, any sequence of nucleotides can be synthesized to cover genic as well as intergenic regions.

Probe size: Early version of cDNA arrays had a probe length of about 200bp. Latest array versions use oligos as short as 70- (Microarrays, Inc.) to 25-mers (Affymetrix). (Feb 2007)

Probe composition: There are tiled and non-tiled DNA arrays. Non-tiled arrays use probes selected according to non-spatial criteria, i.e., the DNA sequences used as probes have no fixed distances in the genome. Tiled arrays, however, select a genomic region (or even a whole genome) and divide it into equal chunks. Such a region is called tiled path. The average distance between each pair of neighboring chunks (measured from the center of each chunk) gives the resolution of the tiled path. A path can be overlapping, end-to-end or spaced. [3]

Array size: The first microarrays used for ChIP-on-Chip contained about 13,000 spotted DNA segments representing all ORFs and intergenic regions from the yeast genome. [2] Nowadays, Affymetrix offers whole-genome tiled yeast arrays with a resolution of 5bp (all in all 3.2 million probes). Tiled arrays for the human genome become more and more powerful, too. Just to name one example, Affymetrix offers a set of seven arrays with about 90 million probes, spanning the complete non-repetitive part of the human genome with about 35bp spacing. (Feb 2007) Besides the actual microarray, other hard- and software equipment is necessary to run ChIP-on-chip experiments. It is generally the case that one company's microarrays can not be analyzed by another company's processing hardware. Hence, buying an array requires also buying the associated workflow equipment. The most important elements are, among others, hybridization ovens, chip scanners, and software packages for subsequent numerical analysis of the raw data.

Workflow of a ChIP-on-chip experiment

Starting with a biological question, a ChIP-on-chip experiment can be divided into three major steps: The first is to set up and design the experiment by selecting the appropriate array and probe type. Second, the actual experiment is performed in the wet-lab. Last, during the dry-lab portion of the cycle, gathered data are analyzed to either answer the initial question or lead to new questions so that the cycle can start again.

Wet-lab portion of the workflow

Workflow overview of the wet-lab portion of a ChIP-on-chip experiment. ChIP-on-chip wet-lab.png
Workflow overview of the wet-lab portion of a ChIP-on-chip experiment.

In the first step, the protein of interest (POI) is cross-linked with the DNA site it binds to in an in vitro environment. Usually this is done by a gentle formaldehyde fixation that is reversible with heat.

Then, the cells are lysed and the DNA is sheared by sonication or using micrococcal nuclease. This results in double-stranded chunks of DNA fragments, normally 1 kb or less in length. Those that were cross-linked to the POI form a POI-DNA complex.

In the next step, only these complexes are filtered out of the set of DNA fragments, using an antibody specific to the POI. The antibodies may be attached to a solid surface, may have a magnetic bead, or some other physical property that allows separation of cross-linked complexes and unbound fragments. This procedure is essentially an immunoprecipitation (IP) of the protein. This can be done either by using a tagged protein with an antibody against the tag (ex. FLAG, HA, c-myc) or with an antibody to the native protein.

The cross-linking of POI-DNA complexes is reversed (usually by heating) and the DNA strands are purified. For the rest of the workflow, the POI is no longer necessary.

After an amplification and denaturation step, the single-stranded DNA fragments are labeled with a fluorescent tag such as Cy5 or Alexa 647.

Finally, the fragments are poured over the surface of the DNA microarray, which is spotted with short, single-stranded sequences that cover the genomic portion of interest. Whenever a labeled fragment "finds" a complementary fragment on the array, they will hybridize and form again a double-stranded DNA fragment.

Dry-lab portion of the workflow

Workflow overview of the dry-lab portion of a ChIP-on-chip experiment. ChIP-on-chip dry-lab.png
Workflow overview of the dry-lab portion of a ChIP-on-chip experiment.

After a sufficiently large time frame to allow hybridization, the array is illuminated with fluorescent light. Those probes on the array that are hybridized to one of the labeled fragments emit a light signal that is captured by a camera. This image contains all raw data for the remaining part of the workflow.

This raw data, encoded as false-color image, needs to be converted to numerical values before the actual analysis can be done. The analysis and information extraction of the raw data often remains the most challenging part for ChIP-on-chip experiments. Problems arise throughout this portion of the workflow, ranging from the initial chip read-out, to suitable methods to subtract background noise, and finally to appropriate algorithms that normalize the data and make it available for subsequent statistical analysis, which then hopefully lead to a better understanding of the biological question that the experiment seeks to address. Furthermore, due to the different array platforms and lack of standardization between them, data storage and exchange is a huge problem. Generally speaking, the data analysis can be divided into three major steps:

During the first step, the captured fluorescence signals from the array are normalized, using control signals derived from the same or a second chip. Such control signals tell which probes on the array were hybridized correctly and which bound nonspecifically.

In the second step, numerical and statistical tests are applied to control data and IP fraction data to identify POI-enriched regions along the genome. The following three methods are used widely: median percentile rank, single-array error, and sliding-window. These methods generally differ in how low-intensity signals are handled, how much background noise is accepted, and which trait for the data is emphasized during the computation. In the recent past, the sliding-window approach seems to be favored and is often described as most powerful.

In the third step, these regions are analyzed further. If, for example, the POI was a transcription factor, such regions would represent its binding sites. Subsequent analysis then may want to infer nucleotide motifs and other patterns to allow functional annotation of the genome. [4]

Strengths and weaknesses

Using tiled arrays, ChIP-on-chip allows for high resolution of genome-wide maps. These maps can determine the binding sites of many DNA-binding proteins like transcription factors and also chromatin modifications.

Although ChIP-on-chip can be a powerful technique in the area of genomics, it is very expensive. Most published studies using ChIP-on-chip repeat their experiments at least three times to ensure biologically meaningful maps. The cost of the DNA microarrays is often a limiting factor to whether a laboratory should proceed with a ChIP-on-chip experiment. Another limitation is the size of DNA fragments that can be achieved. Most ChIP-on-chip protocols utilize sonication as a method of breaking up DNA into small pieces. However, sonication is limited to a minimal fragment size of 200 bp. For higher resolution maps, this limitation should be overcome to achieve smaller fragments, preferably to single nucleosome resolution. As mentioned previously, the statistical analysis of the huge amount of data generated from arrays is a challenge and normalization procedures should aim to minimize artifacts and determine what is really biologically significant. So far, application to mammalian genomes has been a major limitation, for example, due to the significant percentage of the genome that is occupied by repeats. However, as ChIP-on-chip technology advances, high resolution whole mammalian genome maps should become achievable.

Antibodies used for ChIP-on-chip can be an important limiting factor. ChIP-on-chip requires highly specific antibodies that must recognize its epitope in free solution and also under fixed conditions. If it is demonstrated to successfully immunoprecipitate cross-linked chromatin, it is termed "ChIP-grade". Companies that provide ChIP-grade antibodies include Abcam, Cell Signaling Technology, Santa Cruz, and Upstate. To overcome the problem of specificity, the protein of interest can be fused to a tag like FLAG or HA that are recognized by antibodies. An alternative to ChIP-on-chip that does not require antibodies is DamID.

Also available are antibodies against a specific histone modification like H3 tri methyl K4. As mentioned before, the combination of these antibodies and ChIP-on-chip has become extremely powerful in determining whole genome analysis of histone modification patterns and will contribute tremendously to our understanding of the histone code and epigenetics.

A study demonstrating the non-specific nature of DNA binding proteins has been published in PLoS Biology. This indicates that alternate confirmation of functional relevancy is a necessary step in any ChIP-chip experiment. [5]

History

A first ChIP-on-chip experiment was performed in 1999 to analyze the distribution of cohesin along budding yeast chromosome III. [6] Although the genome was not completely represented, the protocol in this study remains equivalent as those used in later studies. The ChIP-on-chip technique using all of the ORFs of the genome (that nevertheless remains incomplete, missing intergenic regions) was then applied successfully in three papers published in 2000 and 2001. [7] [8] [9] The authors identified binding sites for individual transcription factors in the budding yeast Saccharomyces cerevisiae . In 2002, Richard Young's group [10] determined the genome-wide positions of 106 transcription factors using a c-Myc tagging system in yeast. The first demonstration of the mammalian ChIp-on-chip technique reported the isolation of nine chromatin fragments containing weak and strong E2F binding site was done by Peggy Farnham's lab in collaboration with Michael Zhang's lab and published in 2001. [11] This study was followed several months later in a collaboration between the Young lab with the laboratory of Brian Dynlacht which used the ChIP-on-chip technique to show for the first time that E2F targets encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint [12] Other applications for ChIP-on-chip include DNA replication, recombination, and chromatin structure. Since then, ChIP-on-chip has become a powerful tool in determining genome-wide maps of histone modifications and many more transcription factors. ChIP-on-chip in mammalian systems has been difficult due to the large and repetitive genomes. Thus, many studies in mammalian cells have focused on select promoter regions that are predicted to bind transcription factors and have not analyzed the entire genome. However, whole mammalian genome arrays have recently become commercially available from companies like Nimblegen. In the future, as ChIP-on-chip arrays become more and more advanced, high resolution whole genome maps of DNA-binding proteins and chromatin components for mammals will be analyzed in more detail.

Alternatives

Introduced in 2007, ChIP sequencing (ChIP-seq) is a technology that uses chromatin immunoprecipitation to crosslink the proteins of interest to the DNA but then instead of using a micro-array, it uses the more accurate, higher throughput method of sequencing to localize interaction points. [13]

DamID is an alternative method that does not require antibodies.

ChIP-exo uses exonuclease treatment to achieve up to single base pair resolution.

CUT&RUN sequencing uses antibody recognition with targeted enzymatic cleavage to address some technical limitations of ChIP.

Related Research Articles

<span class="mw-page-title-main">DNA-binding protein</span> Proteins that bind with DNA, such as transcription factors, polymerases, nucleases and histones

DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that identify a base pair.

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

In simple words, the cistrome refers a collection of regulatory elements of a set of genes, including transcription factor binding-sites and histone modifications. More specifically, "the set of cis-acting targets of a trans-acting factor on a genome-wide scale, also known as the in vivo genome-wide location of transcription factor binding-sites or histone modifications". The term cistrome is a portmanteau of cistr + ome. The term cistrome was coined by investigators at the Dana–Farber Cancer Institute and Harvard Medical School.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.

<span class="mw-page-title-main">Tiling array</span>

Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface.

<span class="mw-page-title-main">RNA immunoprecipitation chip</span>

RIP-chip is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of interest in vivo. It can also be used to determine relative levels of gene expression, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

Paired-end tags (PET) are the short sequences at the 5’ and 3' ends of a DNA fragment which are unique enough that they (theoretically) exist together only once in a genome, therefore making the sequence of the DNA in between them available upon search or upon further sequencing. Paired-end tags (PET) exist in PET libraries with the intervening DNA absent, that is, a PET "represents" a larger fragment of genomic or cDNA by consisting of a short 5' linker sequence, a short 5' sequence tag, a short 3' sequence tag, and a short 3' linker sequence. It was shown conceptually that 13 base pairs are sufficient to map tags uniquely. However, longer sequences are more practical for mapping reads uniquely. The endonucleases used to produce PETs give longer tags but sequences of 50–100 base pairs would be optimal for both mapping and cost efficiency. After extracting the PETs from many DNA fragments, they are linked (concatenated) together for efficient sequencing. On average, 20–30 tags could be sequenced with the Sanger method, which has a longer read length. Since the tag sequences are short, individual PETs are well suited for next-generation sequencing that has short read lengths and higher throughput. The main advantages of PET sequencing are its reduced cost by sequencing only short fragments, detection of structural variants in the genome, and increased specificity when aligning back to the genome compared to single tags, which involves only one end of the DNA fragment.

Chromatin Interaction Analysis by Paired-End Tag Sequencing is a technique that incorporates chromatin immunoprecipitation (ChIP)-based enrichment, chromatin proximity ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long-range chromatin interactions genome-wide.

<span class="mw-page-title-main">Chromatin immunoprecipitation</span> Genomic technique

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

<span class="mw-page-title-main">ChIP-exo</span>

ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.

Transcription factors are proteins that bind genomic regulatory sites. Identification of genomic regulatory elements is essential for understanding the dynamics of developmental, physiological and pathological processes. Recent advances in chromatin immunoprecipitation followed by sequencing (ChIP-seq) have provided powerful ways to identify genome-wide profiling of DNA-binding proteins and histone modifications. The application of ChIP-seq methods has reliably discovered transcription factor binding sites and histone modification sites.

Selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq) is a technique developed for the rapid identification of DNA binding specificities and affinities of full length monomeric and dimeric transcription factors in a fast and semi-high-throughput fashion.

CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.

H3K9me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 9th lysine residue of the histone H3 protein. H3K9me2 is strongly associated with transcriptional repression. H3K9me2 levels are higher at silent compared to active genes in a 10kb region surrounding the transcriptional start site. H3K9me2 represses gene expression both passively, by prohibiting acetylation as therefore binding of RNA polymerase or its regulatory factors, and actively, by recruiting transcriptional repressors. H3K9me2 has also been found in megabase blocks, termed Large Organised Chromatin K9 domains (LOCKS), which are primarily located within gene-sparse regions but also encompass genic and intergenic intervals. Its synthesis is catalyzed by G9a, G9a-like protein, and PRDM2. H3K9me2 can be removed by a wide range of histone lysine demethylases (KDMs) including KDM1, KDM3, KDM4 and KDM7 family members. H3K9me2 is important for various biological processes including cell lineage commitment, the reprogramming of somatic cells to induced pluripotent stem cells, regulation of the inflammatory response, and addiction to drug use.

CUT&Tag-sequencing, also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA. CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN and CUT&Tag sequencing do not. CUT&Tag sequencing is an improvement over CUT&RUN because it does not require cells to be lysed or chromatin to be fractionated. CUT&RUN is not suitable for single-cell platforms so CUT&Tag is advantageous for these.

ChIL sequencing (ChIL-seq), also known as Chromatin Integration Labeling sequencing, is a method used to analyze protein interactions with DNA. ChIL-sequencing combines antibody-targeted controlled cleavage by Tn5 transposase with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that ChIL-Sequencing does not. ChIL-Seq is a precise technique that reduces sample loss could be applied to single-cells.

<span class="mw-page-title-main">MNase-seq</span> Sk kasid Youtuber

MNase-seq, short for micrococcal nuclease digestion with deep sequencing, is a molecular biological technique that was first pioneered in 2006 to measure nucleosome occupancy in the C. elegans genome, and was subsequently applied to the human genome in 2008. Though, the term ‘MNase-seq’ had not been coined until a year later, in 2009. Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin. DNA bound to histones or other chromatin-bound proteins may remain undigested. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing methods.

References

  1. 1 2 Aparicio, O; Geisberg, JV; Struhl, K (2004). Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. pp. Unit 17.7. doi:10.1002/0471143030.cb1707s23. ISBN   978-0-471-14303-1. ISSN   1934-2616. PMID   18228445. S2CID   30456067.{{cite book}}: |journal= ignored (help)
  2. 1 2 Buck, M. J.; Lieb, J. D. (2004). "ChIP-chip: Considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments". Genomics. 83 (3): 349–360. doi:10.1016/j.ygeno.2003.11.004. PMID   14986705.
  3. ↑ Royce, T. E.; Rozowsky, J. S.; Bertone, P.; Samanta, M.; Stolc, V.; Weissman, S.; Snyder, M.; Gerstein, M. (2005). "Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping". Trends in Genetics. 21 (8): 466–475. doi:10.1016/j.tig.2005.06.007. PMC   1855044 . PMID   15979196.
  4. ↑ "Biomedical Genomics Core - The Research Institute at Nationwide Children's Hospital". genomics.nchresearch.org.
  5. ↑ Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard; Nix, David; Pollard, Daniel A.; Iyer, Venky N.; Hechmer, Aaron; Simirenko, Lisa; Stapleton, Mark; Hendriks, Cris L. Luengo; Chu, Hou Cheng; Ogawa, Nobuo; Inwood, William; Sementchenko, Victor; Beaton, Amy; Weiszmann, Richard; Celniker, Susan E.; Knowles, David W.; Gingeras, Tom; Speed, Terence P.; Eisen, Michael B.; Biggin, Mark D. (2008). "Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm". PLOS Biology. 6 (2): e27. doi:10.1371/journal.pbio.0060027. PMC   2235902 . PMID   18271625.
  6. ↑ Blat, Y.; Kleckner, N. (1999). "Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region". Cell. 98 (2): 249–259. doi: 10.1016/s0092-8674(00)81019-3 . PMID   10428036. S2CID   4689446.
  7. ↑ Lieb, J. D.; Liu, X.; Botstein, D.; Brown, P. O. (2001). "Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association". Nature Genetics. 28 (4): 327–334. doi:10.1038/ng569. PMID   11455386. S2CID   6707334.
  8. ↑ Ren, B.; Robert, F.; Wyrick, J. J.; Aparicio, O.; Jennings, E. G.; Simon, I.; Zeitlinger, J.; Schreiber, J.; Hannett, N.; Kanin, E.; Volkert, T. L.; Wilson, C. J.; Bell, S. P.; Young, R. A. (2000). "Genome-wide location and function of DNA binding proteins". Science. 290 (5500): 2306–2309. Bibcode:2000Sci...290.2306R. doi:10.1126/science.290.5500.2306. PMID   11125145.
  9. ↑ Iyer, V. R.; Horak, C. E.; Scafe, C. S.; Botstein, D.; Snyder, M.; Brown, P. O. (2001). "Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF". Nature. 409 (6819): 533–538. Bibcode:2001Natur.409..533I. doi:10.1038/35054095. PMID   11206552. S2CID   6440664.
  10. ↑ Lee, T. I.; Rinaldi, N. J.; Robert, F.; Odom, D. T.; Bar-Joseph, Z.; Gerber, G. K.; Hannett, N. M.; Harbison, C. T.; Thompson, C. M.; Simon, I.; Zeitlinger, J.; Jennings, E. G.; Murray, H. L.; Gordon, D. B.; Ren, B.; Wyrick, J. J.; Tagne, J. B.; Volkert, T. L.; Fraenkel, E.; Gifford, D. K.; Young, R. A. (2002). "Transcriptional regulatory networks in Saccharomyces cerevisiae". Science. 298 (5594): 799–804. Bibcode:2002Sci...298..799L. doi:10.1126/science.1075090. PMID   12399584. S2CID   4841222.
  11. ↑ Weinmann, A. S.; Bartley, S. M.; Zhang, T.; Zhang, M. Q.; Farnham, P. J. (2001). "Use of chromatin immunoprecipitation to clone novel E2F target promoters". Molecular and Cellular Biology. 21 (20): 6820–6832. doi:10.1128/MCB.21.20.6820-6832.2001. PMC   99859 . PMID   11564866.
  12. ↑ Ren, B.; Cam, H.; Takahashi, Y.; Volkert, T.; Terragni, J.; Young, R. A.; Dynlacht, B. D. (2002). "E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints". Genes & Development. 16 (2): 245–256. doi:10.1101/gad.949802. PMC   155321 . PMID   11799067.
  13. ↑ Johnson, David S.; Mortazavi, Ali; Myers, Richard M.; Wold, Barbara (8 June 2007). "Genome-Wide Mapping of in Vivo Protein-DNA Interactions". Science. 316 (5830): 1497–1502. Bibcode:2007Sci...316.1497J. doi: 10.1126/science.1141319 . PMID   17540862. S2CID   519841.

Further reading

Analysis and software

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