Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1.
Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.
Fitzpatrick
Galina V
GV
Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Pugacheva
Elena M
EM
Shin
Jong-Yeon
JY
Abdullaev
Ziedulla
Z
Yang
Youwen
Y
Khatod
Kavita
K
Lobanenkov
Victor V
VV
Higgins
Michael J
MJ
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D005260:Female; D018392:Genomic Imprinting; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; D022661:RNA, Untranslated; D012097:Repressor Proteins