We have focused attention on the 1.2 kb insulator DNA sequence at the 5 end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. We have shown that this protein is responsible for regulation of imprinted gene expression at several imprinted loci. In order to understand the mechanism of action of CTCF we have continued to extend our earlier studies showing that CTCF molecules interact with co-factors. Among the interactions now under investigation is the interaction of CTCF with the cohesin protein complex, recently shown in the laboratories of Matthias Merkenschlager and others to be essential to the recruitment of cohesin to DNA at some stages of the cell cycle. Our recent results have identified SA2 as the cohesin subunit that directly interacts with CTCF, and also identified the domain of CTCF with which it interacts. We have also used mass spec analysis to detect other co-factors that appear to be important for CTCF insulator function. Using these techniques, we have identified a novel and important co-factor of CTCF, the regulatory protein p68. We have shown that this protein, together with its associated RNA co-factor,binds to CTCF, is present genome-wide at CTCF occupies sites on chromatin, and is essential for insulator activity. We also have evidence that at least one role of p68/SRA is to help stabilize the CTCF/cohesin interaction.
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