Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

Komashko, Vitalina M.; Acevedo, Luis G.; Squazzo, Sharon L.; Iyengar, Sushma; Rabinovich, Alina; O’Geen, Henriette; Green, Roland; Farnham, Peggy J.

doi:10.1101/gr.074609.107

Cited by 44 publications

(58 citation statements)

References 38 publications

(62 reference statements)

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“…Accordingly, recent studies have highlighted the key role of enhancers in cell-type-specific transcriptional regulation (Heintzman et al, 2009;Pennacchio et al, 2006;Pennacchio et al, 2007;Xi et al, 2007). This is in line with the finding that transcriptionally competent promoters are mainly similar in different cell types (Heintzman et al, 2009;Komashko et al, 2008;Xu et al, 2007b). Hence, whereas the chromatin structure at promoters appears mostly conserved in different cell types, the presence of active chromatin marks at enhancers is mainly cell-type-specific and is biased towards enhancers that are in the vicinity of differentially expressed genes.…”

Section: Cell-type-and Context-specific Regulation Of Gene Expressionmentioning

confidence: 52%

Defining specificity of transcription factor regulatory activities

Eeckhoute

Métivier

Salbert

2009

Journal of Cell Science

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the genome, and of chromatin structure and gene expression, have allowed a better understanding of the general rules that underlie the differential activities of a given TF. It has emerged that chromatin structure dictates the differential binding of a given TF to cell-type-specific cis-regulatory elements. The subsequent regulation of TF activity then ensures the functional activation of only the precise subset of all regulatory sites bound by the TF that are required to mediate appropriate gene expression. Ultimately, the organization of the genome within the nucleus, and crosstalk between different cisregulatory regions involved in gene regulation, also participate in establishing a specific transcriptional program. In this Commentary, we discuss how the integration of these different and probably intimately linked regulatory mechanisms allow for TF cell-type-and context-specific modulation of gene expression.Key words: Functional genomics, Transcription factors, Cell-typespecific transcription, Chromatin, Coactivators. Summary Defining specificity of transcription factor regulatory activities Jérôme Eeckhoute*, Raphaël Métivier and Gilles Salbert Journal of Cell Science 4028structure at promoters appears mostly conserved in different cell types, the presence of active chromatin marks at enhancers is mainly cell-type-specific and is biased towards enhancers that are in the vicinity of differentially expressed genes. Indeed, Heintzman and co-workers found that genes expressed in cervical carcinoma HeLa cells and leukaemia K562 cells were mostly identical, and that their promoters exhibited a similar pattern of active histone posttranslational modifications (methylation of lysine 4 and acetylation of lysine 27 of histone H3) (Heintzman et al., 2009). Conversely, these histone marks were found at distinct enhancers in the two cell types, and the distribution of enhancers that had active chromatin features was biased towards the small fraction of those genes that were differentially expressed (Heintzman et al., 2009). Accordingly, TFs that act mainly through binding to enhancers, such as steroid hormone receptors, were shown to exhibit cell-type-specific cistromes (sets of bound regions in the genome) in correlation with differential gene transcriptional regulation (John et al., 2008;Krum et al., 2008;So et al., 2007). Hence, one important function of chromatin is to determine, on a genome-wide scale, the set of regions bound by a given TF in a given cell type.Interestingly, a functional link between chromatin structure, TF recruitment and enhancer activity has been suggested to control the transcriptional regulatory activities exerted by FOXA1 and steroid hormone receptors. FOXA1 is thought to probe the chromatin and to selectively promote the binding of other TFs to regulatory regions of chromatin that were initially condensed; FOXA1 is therefore considered to be a 'pioneer factor' that allows for competency of enhancers (Cirillo et al., 2002;Sekiya et al., 2009;Zaret, 1999). Cell-type-specific FOXA1 activitie...

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Section: Cell-type-and Context-specific Regulation Of Gene Expressionmentioning

confidence: 52%

Defining specificity of transcription factor regulatory activities

Eeckhoute

Métivier

Salbert

2009

Journal of Cell Science

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“…To determine why the 1566 E2F consensus motifs were not bound by E2F1 in MCF7 cells, we examined the results of MCF7 ChIP-chip assays performed using antibodies to E2F4 and E2F6 (Xu et al 2007) and to the silencing marks H3me3K9, H3me3K27, and 5-meC (Komashko et al 2008). We then determined the percentage of the 1566 unbound E2F motifs that were in the top 2000 of each of these datasets.…”

Section: Resultsmentioning

confidence: 99%

“…Characterization of the promoters that contain E2F consensus motifs but are not bound by E2F1 in MCF7 cells. The set of 1566 consensus E2F1 motifs that were unoccupied by E2F1 in MCF7 cells was compared with the top 2000 targets identified in ChIP-chip assays for E2F4, E2F6, H3me3K7, H3me3K9, and 5-meC (Xu et al 2007;Komashko et al 2008). The percentage of promoters in the "unoccupied E2F motif" set that was bound by E2F4 or E2F6, which was found in silenced chromatin (but not bound by E2F4 or E2F6), or was in silenced chromatin and bound by E2F4 or E2F6 is shown.…”

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confidence: 99%

E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

et al. 2008

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We have previously shown that most sites bound by E2F family members in vivo do not contain E2F consensus motifs. However, differences between in vivo target sites that contain or lack a consensus E2F motif have not been explored. To understand how E2F binding specificity is achieved in vivo, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) assays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are (1) the site must be in a core promoter and (2) the region must be utilized as a promoter in that cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including (1) indirect recruitment, (2) looping to the core promoter mediated by an E2F bound to a distal motif, and (3) assisted binding of E2F to a site that weakly resembles an E2F motif. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated fragments. Our findings suggest that in vivo (1) a consensus motif is not sufficient to recruit E2Fs, (2) E2Fs can bind to isolated regions that lack a consensus motif, and (3) binding can require regions other than the best match to the E2F motif.

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“…Retrieval of the crosslinked complexes and analysis of the associated DNA then gave birth to the ChIP assay (14,39,40), which has become ubiquitous in the chromatin field in a multitude of variations (41)(42)(43)(44)(45)(46)(47)(48)(49). Although ChIP assays performed without crosslinking have proven valuable for analyses of stable chromatin complexes (50), crosslinking has made it possible to identify interactions that would not otherwise withstand the isolation procedure.…”

Section: Capture Of Protein-dna Complexesmentioning

confidence: 99%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

330

261

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Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.Prior to its use in the chromatin field, formaldehyde use had a long history in a number of fields, including vaccine production (1, 2) and histology (3). In this review, we focus on its use in chromatin immunoprecipitation approaches and protein-protein interaction studies applied to understand the location and abundance of transcription factor binding along DNA. A recent complementary perspective highlights gaps in knowledge with a particular focus on how formaldehyde crosslinking data have been used to interpret aspects of chromatin three-dimensional organization (4). Here, we briefly review prior work describing formaldehyde reactivity toward proteins, DNA, and their constituent monomers. This information provides a basis for understanding how formaldehyde functions in widely used assays in the chromatin field, and conversely, highlights less well understood aspects of formaldehyde behavior in cells. These issues are of significance for designing crosslinkingbased studies as well as for properly interpreting the resulting data. The analysis of formaldehyde-fixed chromatin has provided fundamental insights into where and when regulatory factors associate with the DNA template in vivo, but it in general does not provide unambiguous information about chromatin binding kinetics. A major goal of ongoing work is to understand kinetic and thermodynamic aspects of chromatin complex assembly at single copy loci in vivo. Development of experimental strategies to achieve these goals will require a deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells.The following discussion provides a framework for understanding aspects of formaldehyde function when used to trap macromolecular complexes in cells, with the main features shown in Fig. 1. Beginning with basic chemical reactivity, this review will explore the ability of formaldehyde to crosslink with proteins and DNA to form protein-protein or protein-DNA complexes, common molecular quenchers, and the potential for crosslink reversal. Progress in capturing crosslinked complexes will also be discussed with an emphasis on the impact of a better understanding of formaldehyde chemistry in vivo. Basic ChemistryFormaldehyde is the smallest aldehyde, an electrophilic molecule susceptible to chemical attack by a wide range of nucleophilic species of ...

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Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

Cited by 44 publications

References 38 publications

Defining specificity of transcription factor regulatory activities

Defining specificity of transcription factor regulatory activities

E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

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