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 β-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.

[1]  M. Nakao,et al.  CTCF-dependent chromatin insulator is linked to epigenetic remodeling. , 2006, Molecular cell.

[2]  Wouter de Laat,et al.  CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. , 2006, Genes & development.

[3]  John T. Lis,et al.  Breaking barriers to transcription elongation , 2006, Nature Reviews Molecular Cell Biology.

[4]  S. Akopov,et al.  Two-dimensional electrophoretic mobility shift assay: identification and mapping of transcription factor CTCF target sequences within an FXYD5-COX7A1 region of human chromosome 19. , 2006, Analytical biochemistry.

[5]  J. León,et al.  Targeting of CTCF to the nucleolus inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism , 2006, Journal of Cell Science.

[6]  C. Chiang,et al.  The General Transcription Machinery and General Cofactors , 2006, Critical reviews in biochemistry and molecular biology.

[7]  T. Oelgeschläger,et al.  Core promoter-selective RNA polymerase II transcription. , 2006, Biochemical Society symposium.

[8]  Guillaume J. Filion,et al.  The Human Enhancer Blocker CTC-binding Factor Interacts with the Transcription Factor Kaiso* , 2005, Journal of Biological Chemistry.

[9]  D. Loukinov,et al.  CTCF binds the proximal exonic region of hTERT and inhibits its transcription , 2005, Nucleic acids research.

[10]  Igor Chernukhin,et al.  CTCF Regulates Growth and Erythroid Differentiation of Human Myeloid Leukemia Cells* , 2005, Journal of Biological Chemistry.

[11]  R. Roeder,et al.  Dynamic regulation of pol II transcription by the mammalian Mediator complex. , 2005, Trends in biochemical sciences.

[12]  E. Klenova,et al.  Regulation of the transcription factor, CTCF, by phosphorylation with protein kinase CK2 , 2005, FEBS letters.

[13]  M. Lerman,et al.  Transcriptional regulator CTCF controls human interleukin 1 receptor-associated kinase 2 promoter. , 2005, Journal of molecular biology.

[14]  R. Ohlsson,et al.  Poly(ADP-ribosyl)ation and Epigenetics: Is CTCF PARt of the Plot? , 2005, Cell cycle.

[15]  Rolf Ohlsson,et al.  Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation , 2004, Nature Genetics.

[16]  D. Butcher,et al.  DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter , 2004, International journal of cancer.

[17]  Rolf Ohlsson,et al.  The binding sites for the chromatin insulator protein CTCF map to DNA methylation-free domains genome-wide. , 2004, Genome research.

[18]  J. Quinn,et al.  YB-1 and CTCF Differentially Regulate the 5-HTT Polymorphic Intron 2 Enhancer Which Predisposes to a Variety of Neurological Disorders , 2004, The Journal of Neuroscience.

[19]  Sylvain V Costes,et al.  Automatic and quantitative measurement of protein-protein colocalization in live cells. , 2004, Biophysical journal.

[20]  V. Pant,et al.  Mutation of a Single CTCF Target Site within the H19 Imprinting Control Region Leads to Loss of Igf2 Imprinting and Complex Patterns of De Novo Methylation upon Maternal Inheritance , 2004, Molecular and Cellular Biology.

[21]  G. Felsenfeld,et al.  CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. , 2004, Molecular cell.

[22]  Kirby D. Johnson,et al.  Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain , 2003, Molecular and Cellular Biology.

[23]  Victor V Lobanenkov,et al.  Thyroid hormone‐regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor , 2003, The EMBO journal.

[24]  C. Kanduri,et al.  The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. , 2003, Genes & development.

[25]  Victor V Lobanenkov,et al.  CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Victor V Lobanenkov,et al.  The novel BORIS + CTCF gene family is uniquely involved in the epigenetics of normal biology and cancer. , 2002, Seminars in cancer biology.

[27]  J. Greenblatt,et al.  Regulation of transcription elongation by phosphorylation. , 2002, Biochimica et biophysica acta.

[28]  J. Norton,et al.  Immunoprecipitation techniques for the analysis of transcription factor complexes. , 2002, Methods.

[29]  R Ohlsson,et al.  CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. , 2001, Trends in genetics : TIG.

[30]  P. Neiman,et al.  Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF. , 2001, Cancer research.

[31]  P. Neiman,et al.  Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF , 2001, Molecular and Cellular Biology.

[32]  R. Tjian,et al.  Orchestrated response: a symphony of transcription factors for gene control. , 2000, Genes & development.

[33]  Victor V Lobanenkov,et al.  Physical and Functional Interaction between Two Pluripotent Proteins, the Y-box DNA/RNA-binding Factor, YB-1, and the Multivalent Zinc Finger Factor, CTCF* , 2000, The Journal of Biological Chemistry.

[34]  Victor V Lobanenkov,et al.  Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive , 2000, Current Biology.

[35]  G. Felsenfeld,et al.  Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene , 2000, Nature.

[36]  A. West,et al.  Structural and functional conservation at the boundaries of the chicken β‐globin domain , 2000 .

[37]  T. Kouzarides,et al.  Transcriptional repression by the insulator protein CTCF involves histone deacetylases. , 2000, Nucleic acids research.

[38]  E. Klenova,et al.  A method of immobilization on the solid support of complex and simple enzymes retaining their activity. , 2000, Analytical biochemistry.

[39]  S. García-Silva,et al.  An Element in the Region Responsible for Premature Termination of Transcription Mediates Repression of c-myc Gene Expression by Thyroid Hormone in Neuroblastoma Cells* , 2000, The Journal of Biological Chemistry.

[40]  A. West,et al.  Structural and functional conservation at the boundaries of the chicken beta-globin domain. , 2000, The EMBO journal.

[41]  C. Allis,et al.  In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment. , 1999, Methods.

[42]  A. West,et al.  The Protein CTCF Is Required for the Enhancer Blocking Activity of Vertebrate Insulators , 1999, Cell.

[43]  G. Felsenfeld,et al.  An insulator element and condensed chromatin region separate the chicken β‐globin locus from an independently regulated erythroid‐specific folate receptor gene , 1999, The EMBO journal.

[44]  A. Bigas,et al.  Differential expression and phosphorylation of CTCF, a c‐myc transcriptional regulator, during differentiation of human myeloid cells , 1999, FEBS letters.

[45]  E. Harlow,et al.  Using Antibodies: A Laboratory Manual , 1999 .

[46]  Shwu‐Yuan Wu,et al.  Immunoaffinity Purification and Functional Characterization of Human Transcription Factor IIH and RNA Polymerase II from Clonal Cell Lines That Conditionally Express Epitope-tagged Subunits of the Multiprotein Complexes* , 1998, The Journal of Biological Chemistry.

[47]  M. Bartolomei,et al.  Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. , 1998, Genes & development.

[48]  L. Vuillard,et al.  Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation. , 1998, European journal of biochemistry.

[49]  Shwu‐Yuan Wu,et al.  TAFII‐independent activation mediated by human TBP in the presence of the positive cofactor PC4 , 1998, The EMBO journal.

[50]  Shwu‐Yuan Wu,et al.  Properties of PC4 and an RNA Polymerase II Complex in Directing Activated and Basal Transcription in Vitro * , 1998, The Journal of Biological Chemistry.

[51]  A. Vostrov,et al.  The zinc finger protein CTCF binds to the APBbeta domain of the amyloid beta-protein precursor promoter. Evidence for a role in transcriptional activation. , 1997, The Journal of biological chemistry.

[52]  J. Acker,et al.  Interactions between the Human RNA Polymerase II Subunits* , 1997, The Journal of Biological Chemistry.

[53]  P. Geyer,et al.  The role of insulator elements in defining domains of gene expression. , 1997, Current opinion in genetics & development.

[54]  Victor V Lobanenkov,et al.  Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF , 1997, Molecular and cellular biology.

[55]  P. Neiman,et al.  An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes , 1996, Molecular and cellular biology.

[56]  M. Dahmus.  Phosphorylation of mammalian RNA polymerase II. , 1996, Methods in enzymology.

[57]  D. Bregman,et al.  Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains , 1995, The Journal of cell biology.

[58]  M. Vigneron,et al.  Synthesis and maturation of viral transcripts in herpes simplex virus type 1 infected HeLa cells: the role of interchromatin granules. , 1995, Gene expression.

[59]  P. Neiman,et al.  CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms , 1993, Molecular and cellular biology.

[60]  M. Groudine,et al.  The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. , 1992, Genes & development.

[61]  D. Eick,et al.  Hold back of RNA polymerase II at the transcription start site mediates down‐regulation of c‐myc in vivo. , 1992, The EMBO journal.

[62]  M. Key,et al.  Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. , 1991, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[63]  R. Ohlsson,et al.  Expression of the human PDGF‐B gene is regulated by both positively and negatively acting cell type‐specific regulatory elements located in the first intron. , 1991, The EMBO journal.

[64]  N. Huskisson,et al.  Induction of differentiation of avian erythroblastosis virus-transformed erythroblasts by the protein kinase inhibitor H7: analysis of the transcription factor EF1. , 1991, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[65]  R. Young,et al.  RNA polymerase II. , 1991, Annual review of biochemistry.

[66]  D. Smith,et al.  Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. , 1988, Gene.

[67]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[68]  T. Graf,et al.  Erythroblast cell lines transformed by a temperature‐sensitive mutant of avian erythroblastosis virus: A model system to study erythroid differentiation in vitro , 1982, Journal of cellular physiology. Supplement.