CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.
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.
Chernukhin
Igor
I
Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom.
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
D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms / Q000473:pathology; D000076246:CCCTC-Binding Factor; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D020014:K562 Cells; D051379:Mice; D041681:NIH 3T3 Cells; D020411:Oligonucleotide Array Sequence Analysis; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D012319:RNA Polymerase II