We have been studying for some years the compound insulator at the 5 end of the chicken beta globin locus. We showed that in addition to its ability to prevent enhancer-promoter interactions, mediated by CTCF, it is also able to block the hetrochromatinization of a reporter gene. This property is independent of CTCF, but we have shown that it does depend on the binding of two other proteins, USF1/2 and BGP1. We have also investigated the properties of BGP1, which binds to separate sites in the insulator. We found that Vezf1, the mouse homolog of BGP1 in mouse ES cells plays an important role in DNA methylation;in its absence methylation levels at critical sites genome-wide are depressed. We made use of an ES cell line in which Vezf1, the mouse BGP1, is deleted. In collaboration with Dr. H. Stuhlmann (Cornell Medical College) we showed earlier that in the absence of Vezf1 the DNA de novo methyl transferase, Dnmt3b, is down regulated, reflecting a decrease in the abundance of the RNA splice variant coding for active Dnmt3b. Wild type phenotype can largely be restored by introducing a Vezf1 expression vector into these cells. We have shown that Vezf1 binds in vivo to a site in an intron of the Dnmt3b gene, and this suggested that Vezf1 might interfere with RNA polymerase II elongation rates, allowing for different alternative splicing pathways. Now we have extended our investigation to a genome-wide survey, in an attempt to understand how Vezf1 suppresses Dnmt3b expression. In these studies, we asked whether Vezf1 may interfere with transcription elongation in such a way as to alter the production of specific splice variants of a gene, as suggested by the experiments described above with mouse ES cells. We find that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1(-/-) mES cells. We now hypothesize that the ability of Vezf1 to slow the passage of RNA pol II is also related to its role as a barrier protein at the chicken beta globin locus. In other work we had shown that the 16 kb heterochromatic region described above is maintained by low levels of transcription and a dicer-dependent mechanism. Vezf1 could block the advance of the polymerase past the 16 kb region, preventing the extension of heterochromatin beyond the barrier insulator.
|Gowher, Humaira; Stuhlmann, Heidi; Felsenfeld, Gary (2008) Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b. Genes Dev 22:2075-84|