ABSTRACT
The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.
Subject(s)
DNA-Binding Proteins/metabolism , Protein Processing, Post-Translational , Repressor Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Amino Acid Sequence , Animals , CCCTC-Binding Factor , Cell Nucleus Structures/metabolism , Cysteine Endopeptidases , DNA/metabolism , DNA-Binding Proteins/chemistry , Endopeptidases/metabolism , HeLa Cells , Humans , Isoenzymes/metabolism , Ligases , Mice , Molecular Sequence Data , Peptide Hydrolases/metabolism , Polycomb-Group Proteins , Promoter Regions, Genetic/genetics , Protein Binding , Protein Transport , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/chemistry , SUMO-1 Protein/metabolism , Ubiquitin-Protein Ligases , Ubiquitins/metabolismABSTRACT
The human 11p15.5 region contains several maternally and paternally imprinted genes. Dysregulation of imprinting of some of these genes occurs in the Beckwith-Wiedemann syndrome and several tumors. Imprinting in this region is controlled by two imprinting control regions (ICR). ICR1 acts as an insulator that regulates the reciprocal imprinting of the IGF2 and H19 genes. A differentially methylated region in ICR2 regulates the expression of a long transcript called KCNQ1OT1. This paternally expressed transcript negatively regulates several paternally imprinted genes around ICR2. Biallelic expression of the KCNQ1OT1 transcript is the primary molecular defect in over 50% of cases of Beckwith-Wiedemann syndrome. To understand the role of KCNQ1OT1 in regulating ICR2 we characterized its promoter. The critical promoter is approximately 300 bp and it is surrounded by inhibitory elements within the CpG island. The promoter activity is strongly inhibited by cytosine methylation in keeping with the finding that the inactive maternal promoter is methylated in vivo. We have identified the transcription start sites and four CCAAT boxes upstream of the 5'-most start site. Mutation of the CCAAT boxes produced impairment of promoter activity. Transfection and gel mobility shift experiments suggest that binding of the factor NF-Y to the CCAAT boxes is important for promoter activity.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Genomic Imprinting/genetics , Membrane Proteins/genetics , Potassium Channels, Voltage-Gated/genetics , Promoter Regions, Genetic/genetics , Base Sequence , CCAAT-Binding Factor/metabolism , Cell Line , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Methylation , Genes, Reporter/genetics , Humans , Molecular Sequence Data , Mutagenesis, Insertional , Mutation/genetics , Protein Binding , Response Elements/genetics , Transcription Initiation SiteABSTRACT
The imprinting of the genes on human chromosome 11p15.5 is thought to be controlled by two imprinting control regions located in two differentially methylated CpG islands upstream of the H19 gene (H19 DMR) and in intron 10 of the KCNQ1 gene (KvDMR). We have examined sequences in the human 11p15.5 genomic imprinted region for the presence of insulators and silencers using a position- and enhancer-dependent stable transfection assay. We have confirmed the existence of insulators in H19 DMR and discovered two novel insulators in the IGF2 gene. We have also found two novel silencer sequences; one is located in KvDMR, a region that is thought to contain the promoter for the KCNQ1OT1 transcript, and another is in the CDKN1C gene. We have demonstrated binding of CTCF protein in vitro to all the insulator and silencer sequences that we have detected. We discuss the differences in the regulation of imprinting controlled by the two imprinting control regions in chromosome 11p.
