Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation.
Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.
Farrar
Dawn
D
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom.
Rai
Sushma
S
Chernukhin
Igor
I
Jagodic
Maja
M
Ito
Yoko
Y
Yammine
Samer
S
Ohlsson
Rolf
R
Murrell
Adele
A
Klenova
Elena
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D000076246:CCCTC-Binding Factor; D002460:Cell Line; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D018392:Genomic Imprinting; D006367:HeLa Cells; D006801:Humans; D006822:Hybrid Cells; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D017931:DNA Primers; C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D011994:Recombinant Proteins; D012097:Repressor Proteins; D011064:Poly Adenosine Diphosphate Ribose; D007335:Insulin-Like Growth Factor II; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases