Dataset Information

CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

ABSTRACT: CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

SUBMITTER: Chernukhin I

PROVIDER: S-EPMC1820452 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

Chernukhin Igor I Shamsuddin Shaharum S Kang Sung Yun SY Bergström Rosita R Kwon Yoo-Wook YW Yu Wenqiang W Whitehead Joanne J Mukhopadhyay Rituparna R Docquier France F Farrar Dawn D Morrison Ian I Vigneron Marc M Wu Shwu-Yuan SY Chiang Cheng-Ming CM Loukinov Dmitri D Lobanenkov Victor V Ohlsson Rolf R Klenova Elena E

Molecular and cellular biology 20070108 5

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit o ...[more]

PMID: 17210645

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Project description:Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial ? subunit and forms a complex with Rpb3 subunit analogous to prokaryotic ?2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11b?, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11b?. We demonstrated that the hRPB11b? interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11b? resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11b? isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3-ATF4 (not hRPB11a-ATF4) and hRpb11b?-ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11b? and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of ?-like subunits (Rpb3-Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11-ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

Dataset Information

CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

Publications

CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

Similar Datasets

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