Chromatin Immunoprecipitation Assay to Analyze the Effect of G-Quadruplex Interactive Agents on the Binding of RNA Polymerase II and Transcription Factors to a Target Promoter Region

2019 ◽  
pp. 233-242
Author(s):  
Daekyu Sun
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Promoter Region ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
G Quadruplex ◽  
Target Promoter

scholarly journals Uniform Distribution of Elongating RNA Polymerase II Complexes in Transcribed Gene Locus

2011 ◽  
Vol 286 (27) ◽  
pp. 23817-23822 ◽  
Author(s):  
Kadri Peil ◽  
Signe Värv ◽  
Marko Lõoke ◽  
Kersti Kristjuhan ◽  
Arnold Kristjuhan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uniform Distribution ◽  
Cell Population ◽  
Chromatin Immunoprecipitation ◽  
Cell Level ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Genome Wide ◽  
Dna Fragment

The intensity of gene transcription is generally reflected by the level of RNA polymerase II (RNAPII) recruitment to the gene. However, genome-wide studies of polymerase occupancy indicate that RNAPII distribution varies among genes. In some loci more polymerases are found in the 5′ region, whereas in other loci, in the 3′ region of the gene. We studied the distribution of elongating RNAPII complexes at highly transcribed GAL-VPS13 locus in Saccharomyces cerevisiae and found that in the cell population the amount of polymerases gradually decreased toward the 3′ end of the gene. However, the conventional chromatin immunoprecipitation assay averages the signal from the cell population, and no data on single cell level can be gathered. To study the spacing of elongating polymerases on single chromosomes, we used a sequential chromatin immunoprecipitation assay for the detection of multiple RNAPII complexes on the same DNA fragment. Our results demonstrate uniform distribution of elongating polymerases throughout all regions of the GAL-VPS13 gene.

scholarly journals Binding of estrogen receptor α/β heterodimers to chromatin in MCF-7 cells

2009 ◽  
Vol 43 (2) ◽  
pp. 65-72 ◽  
Author(s):  
Zoi Papoutsi ◽  
Chunyan Zhao ◽  
Milica Putnik ◽  
Jan-Åke Gustafsson ◽  
Karin Dahlman-Wright
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Promoter Region ◽  
Estrogen Receptor Α ◽  
Chromatin Immunoprecipitation Assay ◽  
Regulate Gene Expression ◽  
Immunoprecipitation Assay ◽  
Binding Regions ◽  
Mcf 7

Estrogen receptors (ERs), ERα and ERβ, belong to a group of transcription factors that, upon ligand binding, regulate gene expression by binding to specific DNA regions in chromatin as dimers. In this article, we applied the sequential chromatin immunoprecipitation assay (Re-ChIP) to study the simultaneous presence of ERα and ERβ on various DNA-binding regions in intact chromatin. ERα/β heterodimers were isolated by precipitation with anti-ERβ antibody followed by anti-ERα antibody from a stable MCF-7-derived cell line that expresses endogenous ERα and an inducible version of ERβ. The Re-ChIP method was first validated based on the detection of ERα/β heterodimers bound to a promoter region of the pS2 gene known to bind both ERα and ERβ. We next examined 12 ER-binding sites using Re-ChIP assays for ERα/β heterodimer recruitment. Our results confirmed the recruitment of ERα/β heterodimers to all these regions. This study represents the first demonstration of binding of ERα/β heterodimers to various DNA-binding regions in intact chromatin.

scholarly journals Disruption of cardiac Med1 inhibits RNA polymerase II promoter occupancy and promotes chromatin remodeling

2019 ◽  
Vol 316 (2) ◽  
pp. H314-H325 ◽  
Author(s):  
Duane D. Hall ◽  
Kathryn M. Spitler ◽  
Chad E. Grueter
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Chromatin Accessibility ◽  
Gene Promoters ◽  
Specific Expression ◽  
Pol Ii ◽  
Transcriptional Start Sites

The Mediator coactivator complex directs gene-specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA polymerase II (Pol II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. Previously, we found that deletion of Med1 in cardiomyocytes deregulates more than 5,000 genes and promotes acute heart failure. Therefore, we hypothesized that Med1 deficiency disrupts enhancer-promoter coupling. Using chromatin immunoprecipitation-coupled deep sequencing (ChIP-seq; n = 3/ChIP assay), we found that the Pol II pausing index is increased in Med1 knockout versus floxed control mouse hearts primarily due to a decrease in Pol II occupancy at the majority of transcriptional start sites without a corresponding increase in elongating species. Parallel ChIP-seq assays reveal that Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation, which is indicative of open and active chromatin at transcriptional start sites, whereas H3 K27 trimethylated levels, representing condensed and repressed DNA, are broadly increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated superenhancer regions and their enriched transcription factor-binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart and supports the proposed role of Mediator in establishing preinitiation complex formation. We also found that Med1 determines chromatin accessibility within genes and enhancer regions and propose that the composition of transcription factors associated with superenhancer changes to direct gene-specific expression. NEW & NOTEWORTHY Based on our previous findings that transcriptional homeostasis and cardiac function are disturbed by cardiomyocyte deletion of the Mediator coactivator Med1 subunit, we investigated potential underlying changes in RNA polymerase II localization and global chromatin accessibility. Using chromatin immunoprecipitation sequencing, we found that disrupted transcription arises from a deficit in RNA polymerase II recruitment to gene promoters. Furthermore, active versus repressive chromatin marks are redistributed within gene loci and at enhancer regions correlated with gene expression changes.

scholarly journals Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

1997 ◽  
Vol 110 (15) ◽  
pp. 1781-1791 ◽  
Author(s):  
M.A. Grande ◽  
I. van der Kraan ◽  
L. de Jong ◽  
R. van Driel
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Glucocorticoid Receptors ◽  
Cultured Cells ◽  
Spatial Relationship ◽  
Immunofluorescence Labelling ◽  
Transcription Sites ◽  
Nuclear Domains

We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogeneously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 microm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.

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