scholarly journals Deconvolution of Chromatin Immunoprecipitation-Microarray (ChIP-chip) Analysis of MBF Occupancies Reveals the Temporal Recruitment of Rep2 at the MBF Target Genes

2010 ◽  
Vol 10 (1) ◽  
pp. 130-141 ◽  
Author(s):  
Majid Eshaghi ◽  
Lei Zhu ◽  
Zhaoqing Chu ◽  
Juntao Li ◽  
Chee Seng Chan ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Time Course ◽  
Target Genes ◽  
S Phase ◽  
Deconvolution Analysis ◽  
Microarray Chip ◽  
Rnap Ii ◽  
Chip Chip ◽  
Chip Analysis

ABSTRACT MBF (or DSC1) is known to regulate transcription of a set of G 1 /S-phase genes encoding proteins involved in regulation of DNA replication. Previous studies have shown that MBF binds not only the promoter of G 1 /S-phase genes, but also the constitutive genes; however, it was unclear if the MBF bindings at the G 1 /S-phase and constitutive genes were mechanistically distinguishable. Here, we report a chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF binding in the Schizosaccharomyces pombe genome using high-resolution genome tiling microarrays. ChIP-chip analysis indicates that the majority of the MBF occupancies are located at the intragenic regions. Deconvolution analysis using Rpb1 ChIP-chip results distinguishes the Cdc10 bindings at the Rpb1-poor loci (promoters) from those at the Rpb1-rich loci (intragenic sequences). Importantly, Res1 binding at the Rpb1-poor loci, but not at the Rpb1-rich loci, is dependent on the Cdc10 function, suggesting a distinct binding mechanism. Most Cdc10 promoter bindings at the Rpb1-poor loci are associated with the G 1 /S-phase genes. While Res1 or Res2 is found at both the Cdc10 promoter and intragenic binding sites, Rep2 appears to be absent at the Cdc10 promoter binding sites but present at the intragenic sites. Time course ChIP-chip analysis demonstrates that Rep2 is temporally accumulated at the coding region of the MBF target genes, resembling the RNAP-II occupancies. Taken together, our results show that deconvolution analysis of Cdc10 occupancies refines the functional subset of genomic binding sites. We propose that the MBF activator Rep2 plays a role in mediating the cell cycle-specific transcription through the recruitment of RNAP-II to the MBF-bound G 1 /S-phase genes.

scholarly journals Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

2005 ◽  
Vol 187 (17) ◽  
pp. 6166-6174 ◽  
Author(s):  
Christopher D. Herring ◽  
Marni Raffaelle ◽  
Timothy E. Allen ◽  
Elenita I. Kanin ◽  
Robert Landick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Reading Frame ◽  
E Coli ◽  
Genome Wide ◽  
Polymerase Binding ◽  
Chip Chip ◽  
Negative Detection

ABSTRACT The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

Genome Wide Approaches to Identify Protein-DNA Interactions

2020 ◽  
Vol 26 (42) ◽  
pp. 7641-7654 ◽  
Author(s):  
Tao Ma ◽  
Zhenqing Ye ◽  
Liguo Wang
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Target Genes ◽  
Rapid Development ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Computational Approaches ◽  
Factor Binding ◽  
Genome Wide ◽  
Chip Chip

Background: Transcription factors are DNA-binding proteins that play key roles in many fundamental biological processes. Unraveling their interactions with DNA is essential to identify their target genes and understand the regulatory network. Genome-wide identification of their binding sites became feasible thanks to recent progress in experimental and computational approaches. ChIP-chip, ChIP-seq, and ChIP-exo are three widely used techniques to demarcate genome-wide transcription factor binding sites. Objective: This review aims to provide an overview of these three techniques including their experiment procedures, computational approaches, and popular analytic tools. Conclusion: ChIP-chip, ChIP-seq, and ChIP-exo have been the major techniques to study genome- wide in vivo protein-DNA interaction. Due to the rapid development of next-generation sequencing technology, array-based ChIP-chip is deprecated and ChIP-seq has become the most widely used technique to identify transcription factor binding sites in genome-wide. The newly developed ChIP-exo further improves the spatial resolution to single nucleotide. Numerous tools have been developed to analyze ChIP-chip, ChIP-seq and ChIP-exo data. However, different programs may employ different mechanisms or underlying algorithms thus each will inherently include its own set of statistical assumption and bias. So choosing the most appropriate analytic program for a given experiment needs careful considerations. Moreover, most programs only have command line interface so their installation and usage will require basic computation expertise in Unix/Linux.

Hoxa9 and Meis1 Bind Highly Conserved Elements Near Targets Regulated in Leukemia Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3362-3362
Author(s):  
Kajal V. Sitwala ◽  
Yongsheng Huang ◽  
Monisha Dandekar ◽  
Gordon Robertson ◽  
Timothee Cezard ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Expression System ◽  
Leukemic Cells ◽  
P Value ◽  
Intergenic Sequence ◽  
Binding Motif ◽  
Chip Chip

Abstract The homeobox transcription factors Hoxa9 and Meis1 are critical mediators of transformation by MLL fusion proteins and have also been associated with poor prognosis in other AML subtypes. Despite their pivotal role in leukemia, their mechanism of cooperation and their direct target genes are largely unknown. We have established an experimental model to address these questions by transforming murine hematopoietic progenitors with epitope-tagged forms of Hoxa9 and Meis1. Chromatin immunoprecipitation and massively parallel Illumina sequencing (ChIP-seq) was used to identify binding sites for Hoxa9 and Meis1 across the genome in leukemic cells. We generated 1.7 Gb of sequence data for immunoprecipitated DNA and an untagged control, using two biological replicates, and filtered out (1) regions with nonspecific binding, (2) enriched regions that overlapped certain types of repetitive elements, and (3) nonreproducible enriched regions. We validated a subset of enriched regions by ChIP-chip and ChIP-PCR. De novo motif discovery verified that the resulting set of regions contained a motif that corresponded (E value = 6E-16) to a published Hoxa9 binding motif (Shen et al.; Mol. Cell. Biol. 1999, 19:3051–61). Overlapping or immediately adjacent binding of Hoxa9 and Meis1 was seen for 52% of Hoxa9 and 33% of Meis1 enriched regions, suggesting that these two transcription factors cooperate directly in oncogenesis rather than acting through parallel pathways. Only 7% of enriched regions fell within 2 Kb of known transcriptional start sites, while 46% of regions were within gene boundaries; the remaining peaks were intergenic. Remarkably, given the low overall level of intergenic sequence conservation, over 95% of enriched regions overlapped evolutionarily highly conserved regions (Phastcons for 17 mammalian species >50%; P value<0.0001 [compared to untagged control peaks]). Our experiments suggest that targets of Hoxa9 and Meis1 in both hematopoiesis and leukemic transformation are regulated in part by highly conserved non-coding elements (HCNE), which are recently recognized genomic elements whose functions remain elusive and which are typically absent from widely used promoter microarrays. RNA expression analysis by our group and others confirms that Hoxa9 and Meis1 influence a large number of downstream targets. We used an inducible Hoxa9 expression system to identify genes that were differentially expressed at 72, 96, and 120 h after Hoxa9 withdrawal; 753, 1116, and 2975 transcripts showed >2-fold change, respectively. Phenotypic changes are evident by 96 h; therefore, the 72 h timepoint is most informative of direct Hoxa9 targets. We compared these data to genes containing ChIP-seq peaks and genes with transcription start sites closest to intergenic peaks. ~10% of genes altered at 72 h were associated with a ChIP-seq peak. Another group of investigators identified targets of Meis1 through comparison of Hoxa9- and Hoxa9+Meis1-immortalized cells (Wang et al.; Blood 2005, 106:254–63); ~14% of reported Meis1 targets were associated with a ChIP-seq peak. Hoxa9 and Meis1 binding sites in selected target genes, such the tyrosine kinase Flt3 and the surface marker Cd34, have been confirmed by ChIP-PCR/ChIP-chip. Our current data support a model in which Hoxa9 and Meis1 directly modulate a substantial number of downstream effectors through co-occupancy of regulatory motifs within proximal promoters, intragenic elements, and distally located regions. Studies are underway to determine whether Hoxa9/Meis1 binding influences gene expression and epigenetic modification at promoters and particularly at HCNEs.

scholarly journals GATA-1 binding sites mapped in the  -globin locus by using mammalian chIp-chip analysis

2002 ◽  
Vol 99 (5) ◽  
pp. 2924-2929 ◽  
Author(s):  
C. E. Horak ◽  
M. C. Mahajan ◽  
N. M. Luscombe ◽  
M. Gerstein ◽  
S. M. Weissman ◽  
...  
Keyword(s):  
Binding Sites ◽  
Chip Chip ◽  
Chip Analysis

scholarly journals Chromatin Immunoprecipitation on Microarray Analysis of Smad2/3 Binding Sites Reveals Roles of ETS1 and TFAP2A in Transforming Growth Factor β Signaling

2008 ◽  
Vol 29 (1) ◽  
pp. 172-186 ◽  
Author(s):  
Daizo Koinuma ◽  
Shuichi Tsutsumi ◽  
Naoko Kamimura ◽  
Hirokazu Taniguchi ◽  
Keiji Miyazawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Hacat Keratinocytes ◽  
Small Interfering Rnas ◽  
Promoter Regions ◽  
Essential Resource

ABSTRACT The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor β (TGF-β) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-β-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-β-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-β-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-β and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.

scholarly journals Multimodal Regulation of E2F1 Gene Expression by Progestins

2010 ◽  
Vol 30 (8) ◽  
pp. 1866-1877 ◽  
Author(s):  
Hilary E. Wade ◽  
Sakiko Kobayashi ◽  
Matthew L. Eaton ◽  
Michelle S. Jansen ◽  
Edward K. Lobenhofer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Feedback Loop ◽  
Target Gene ◽  
Target Genes ◽  
New Paradigm ◽  
Regulatory Pathways ◽  
Maximal Induction ◽  
Chip Analysis ◽  
E2f1 Expression

ABSTRACT An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.

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