automating-chip-seq-experiments-to-generate-epigenetic-profiles-on

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.

The Paf1 complex physically and functionally associates with transcription elongation factors in vivo.

Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.

Exploring functional relationships between components of the gene expression machinery.

Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation.

Suz12 binds to silenced regions of the genome in a cell-type-specific manner.

Advances in high-throughput technologies, such as ChIP-chip, and the completion of human and mouse genomic sequences now allow analysis of the mechanisms of gene regulation on a systems level. In this study, we have developed a computational genomics approach (termed ChIPModules), which begins with experimentally determined binding sites and integrates positional weight matrices constructed from transcription factor binding sites, a comparative genomics approach, and statistical learning methods to identify transcriptional regulatory modules. We began with E2F1 binding site information obtained from ChIP-chip analyses of ENCODE regions, from both HeLa and MCF7 cells. Our approach not only distinguished targets from nontargets with a high specificity, but it also identified five regulatory modules for E2F1. One of the identified modules predicted a colocalization of E2F1 and AP-2alpha on a set of target promoters with an intersite distance of <270 bp. We tested this prediction using ChIP-chip assays with arrays containing approximately 14,000 human promoters. We found that both E2F1 and AP-2alpha bind within the predicted distance to a large number of human promoters, demonstrating the strength of our sequence-based, unbiased, and universal protocol. Finally, we have used our ChIPModules approach to develop a database that includes thousands of computationally identified and/or experimentally verified E2F1 target promoters.

A computational genomics approach to identify cis-regulatory modules from chromatin immunoprecipitation microarray data--a case study using E2F1.

We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing KrÃ¼ppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1.

Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs.

Wir f&uuml;hrten eine Genom-Skala Chromatin Immunopr&auml;zipitation (ChIP)-Vergleich der beiden &Auml;nderungen (Trimethylation von Lysin 9 [H3me3K9] und Trimethylation von Lysin 27 [H3me3K27]) von Histon H3 in Ntera2 Hoden-Karzinom-Zellen und in drei verschiedenen anatomischen Quellen der prim&auml;ren menschlichen Fibroblasten-chip. Wir fanden, dass die beiden &Auml;nderungen in jedem der Zelltypen differentiell an die F&ouml;rderer der beiden gr&ouml;&szlig;ten Klassen von Transkriptionsfaktoren bereichert wurden. Insbesondere Zink-Finger (ZNF)-Gene wurden durch H3me3K9 gebunden und Hox-Gene wurden durch H3me3K27 gebunden. Wir haben zuvor gezeigt, dass die Polycomb repressiven komplexe 2 zust&auml;ndig f&uuml;r die Vermittlung von Trimethylation von Lysin 27 von Histon H3 in menschlichen Krebszellen. Im Gegensatz dazu gibt es wenig &Uuml;berschneidungen zwischen H3me3K9 Ziele und Baugruppen f&uuml;r die Polycomb repressiven Komplex 2, darauf hindeutet, dass eine unterschiedliche Histon-Methyltransferase verantwortlich f&uuml;r die H3me3K9-&Auml;nderung ist. Fr&uuml;here Studien zeigten, dass SETDB1 Trimethylate H3 auf Lysin 9, k&ouml;nnen mithilfe von in-vitro- oder k&uuml;nstliche Anbindehaltung Proben. SETDB1 ist gedacht, um gegen Chromatin durch komplexe mit den KAP1 Corepressor rekrutiert werden. Um festzustellen, ob ein KAP1-haltige Komplex Trimethylation der identifizierten H3me3K9 Ziele vermittelt, wir ChIP-Chip-Tests durchgef&uuml;hrt und identifiziert KAP1 Zielgene menschlichen 5-kb-Projekttr&auml;ger Arrays verwenden. Wir fanden, dass eine gro&szlig;e Anzahl von Genen des ZNF Transkriptionsfaktoren KAP1 und H3me3K9 in Normal und Krebs Zellen gebunden waren. Um unsere Studien KAP1 zu erweitern, f&uuml;hrten wir als n&auml;chstes eine vollst&auml;ndige genomische Analyse KAP1 Bindung mit einem 38-Array Fliesen, ~ 7.000 KAP1 Bindungsstellen zu identifizieren. Die identifizierten KAP1 Ziele wurden hochangereichertes f&uuml;r C2H2 ZNFs, vor allem diejenigen, die Kr&uuml;ppel-assoziierte Feld (KRABBE) Dom&auml;nen enthalten. Obwohl die meisten KAP1 Bindungsstellen innerhalb Kernregionen Projekttr&auml;ger waren, wurden die Bindungsstellen in der N&auml;he von ZNF Gene Interessanterweise erheblich innerhalb der transkribierten Regionen der Zielgene bereichert. Da KAP1 an die DNA durch Interaktion mit der KRABBE-ZNF Proteine eingestellt ist, wird empfohlen, die Expression von Genen KRAB-ZNF &uuml;ber eine Auto-regulatorischen Mechanismus KAP1 gesteuert werden kann.

Genomweiten Analyse KAP1 Bindung schl&auml;gt &uuml;berl&auml;sst der KRAB-ZNFs.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

Wir berichten, die Erstellung und Analyse von funktionalen Daten aus mehreren unterschiedlichen Experimente durchgef&uuml;hrt auf einem gezielten 1 % des menschlichen Genoms im Rahmen der Pilotphase des Projektes ENCODE. Diese Daten wurden weiter integriert und erg&auml;nzt durch eine Reihe von evolution&auml;ren und computergest&uuml;tzte Analysen. Unsere Ergebnisse voraus zusammen, das kollektive Wissen &uuml;ber menschliche Genom-Funktion in einigen wichtigen Bereichen. Unsere Studien geben Sie zuerst &uuml;berzeugenden Beweis, dass das Genom durchdringend transkribiert wird, so dass die meisten ihrer Grundlagen kann, in prim&auml;ren Abschriften gefunden werden, einschlie&szlig;lich nicht-Protein-kodierenden Abschriften und solche, die weitgehend &uuml;berlappen. Zweite, systematische Untersuchung der Transkriptionsregulation hat neues Verst&auml;ndnis &uuml;ber Transkription Start-Websites, einschlie&szlig;lich ihrer Beziehung zu bestimmten regulatorische Sequenzen des Chromatin-Zug&auml;nglichkeit und Histon-&Auml;nderung ergeben. Drittens eine anspruchsvollere Ansicht von Chromatin Struktur hat sich entwickelt, einschlie&szlig;lich ihrer Wechselwirkung mit der DNA-Replikation und transkriptionelle Regulation. Schlie&szlig;lich f&uuml;hrte Integration dieser neuen Quellen von Informationen, insbesondere in Bezug auf S&auml;ugetier-Entwicklung basierend auf inter - und Intra-Spezies Sequenz vergleichen, mechanistischen und evolution&auml;re Erkenntnisse &uuml;ber die funktionale Landschaft des menschlichen Genoms. Zusammen sind diese Studien einen Pfad f&uuml;r die Aus&uuml;bung der eine umfassendere Charakterisierung des menschlichen Genoms Funktion definieren.

Ermittlung und Analyse der Funktionselemente in 1 % des menschlichen Genoms durch das ENCODE-Pilotprojekt.

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.

Noncoding RNAs (NcRNA) werden an epigenetischen Verordnung teilnehmen allerdings schlecht verstanden. Hier wir charakterisieren die transkriptionelle Landschaft der vier menschlichen HOX-Loci auf f&uuml;nf paar Aufl&ouml;sung in 11 anatomisch Websites und 231 HOX-NcRNAs, die bekannte transkribierte Regionen von mehr als 30 Kilobases erweitern zu identifizieren. HOX NcRNAs werden r&auml;umlich entlang Entwicklungsst&ouml;rungen Achsen ausgedr&uuml;ckt und besitzen eindeutige Sequenz Motive und ihren Ausdruck abgrenzt Breite chromosomale Dom&auml;nen der differenziellen Histon-Methylierung und der RNS-Polymerase Zug&auml;nglichkeit. Wir identifiziert ein 2.2 Kilobase NcRNA wohnten in der HOXC-Locus bezeichnet, HEI&szlig;LUFTBALLON, die Transkription in Trans &uuml;ber 40 Kilobases der HOXD Locus unterdr&uuml;ckt. HOTAIR interagiert mit Polycomb repressiven Complex 2 (PRC2) und f&uuml;r PRC2 Belegung und Histon H3 Lysin-27 Trimethylation HOXD Locus ist erforderlich. Somit kann Transkription von NcRNA chromosomale Dom&auml;nen der Gen-silencing Fernweg abgrenzen; Diese Ergebnisse haben gro&szlig;e Auswirkungen auf Genregulation in Entwicklung und Krankheitzust&auml;nden.

Funktionale Abgrenzung der aktiven und leisen Chromatin Dom&auml;nen in menschlichen HOX-Loci von noncoding RNAs.

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.

Article	Year
The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. The EMBO journal\| PubMed ID: 11927560	2002
Exploring functional relationships between components of the gene expression machinery. Nature structural & molecular biology\| PubMed ID: 15702072	2005
Suz12 binds to silenced regions of the genome in a cell-type-specific manner. Genome research\| PubMed ID: 16751344	2006
A computational genomics approach to identify cis-regulatory modules from chromatin immunoprecipitation microarray data--a case study using E2F1. Genome research\| PubMed ID: 17053090	2006
Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs. PLoS genetics\| PubMed ID: 17542650	2007
Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature\| PubMed ID: 17571346	2007
Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell\| PubMed ID: 17604720	2007
Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells. Genome research\| PubMed ID: 18347325	2008

Sharon Squazzo

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