Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3

Abstract

The 5'-ends of two paternally expressed mouse genes, Peg3 and Usp29, are jointly associated with a CpG island that exhibits allele-specific methylation. Sequence comparison of the regions derived from human, mouse and cow revealed the presence of two evolutionarily conserved sequence motifs including one that is repeated multiple times within the first intron of Peg3 in all three mammals. DNA mobility shift and chromatin immunoprecipitation (ChIP) assays clearly demonstrated that this motif is an in vivo binding site for the Gli-type transcription factor YY1. The YY1-binding site contains one CpG dinucleotide, and methylation of this CpG site abolishes the binding activity of YY1 in vitro. The Peg3 YY1-binding sites are methylated only on the maternal chromosome in vivo, and ChIP assays confirmed that YY1 binds specifically to the paternal allele of the gene. Promoter, enhancer and insulator assays with deletion constructs of sequence surrounding the YY1-binding sites indicate that the region functions as a methylation-sensitive insulator that may influence the imprinted expression of Peg3 and neighboring genes. The current study is the first report demonstrating the involvement of YY1 in methylation-sensitive insulator activity and suggests a potential role of this highly conserved protein in mammalian genomic imprinting.

A prominent and conserved role for YY1 in Xist transcriptional activation

“…As a control, we show that both methylated and unmethylated Xist alleles can be recovered in ChIP experiment targeting histone H3. These results confirm the allelic binding of YY1 to the active Xist promoter and are in agreement with previous reports of DNA methylation-sensitive binding of YY1 45-47 .…”

Characterization of the ICCE Repeat in Mammals Reveals an Evolutionary Relationship with the DXZ4 Macrosatellite through Conserved CTCF Binding Motifs

“…2000; Filippova et al. 2001) and YY1 (Gaston and Fried 1995; Kim et al. 2003), if these YY1 motifs are occupied, we would predict that this would be Xi-specific.…”

Retroposition and evolution of the DNA-binding motifs of YY1, YY2 and REX1

“…In the case of YY1, we have used the same set of duplex oligonucleotides used in a previous study to demonstrate the subtle but unique property of YY1, methylation-sensitive DNA-binding (5). As expected, the DNA-binding domain, as part of the GST-YY1 fusion protein, showed an almost identical pattern of DNA binding as endogenous YY1 protein (Figure 6A).…”

“…Identified DNA-binding motifs were further confirmed with gel shift assays using three fusion proteins. For the gel shift assays of the YY1 and YY2 fusion proteins, we have used a set of the six different duplex probes that have been previously used for testing the methylation-sensitive DNA-binding activity of endogenous YY1 (5). Four different probes have an identical sequence as the CSE2 probe containing one YY1 binding site indicated by an underline.…”