So he decided to work directly with chromatin. The move resulted in a big change for the longtime chemist. "The thing with chromatin," he says, "is that I got sucked into the biology and trying to figure out what is the biological function of this DNA-protein packaging."
Felsenfeld's early work in chromatin biology was aimed at understanding how a gene is controlled through a combination of histone interactions and transcription factors. He used the four-gene chicken beta-globin gene locus for his studies, which was an ideal system for his work, because chicken blood cells have stable chromatin that can be isolated easily in large quantities. Using the beta-globin model, his group contributed numerous findings regarding the role of structural and biochemical changes in chromatin in regulating globin gene expression.
Later, Felsenfeld became more intrigued at what lay at the edges of the beta-globin locus. In blood cells, the globin locus is an open and accessible chromatin domain; at the terminus, where the locus borders a stretch of condensed chromatin, there is a DNase hypersensitive site that appears to mark the boundary. (HS sites are short regions of chromatin distinguished by their extreme sensitivity to nuclease cleavage.)
Felsenfeld proposed testing this region to determine if it did, in fact, constitute a boundary between the open and closed domains. At the time, there was only one published example of such an insulator element, the gypsy element in Drosophila. Victor Corces and Dale Dorsett had shown that gypsy could block an enhancer's ability to increase gene promoter activity if positioned between the two, effectively insulating the promoter from enhancer influence. Felsenfeld's group tested the HS site and found that it behaved similarly.
They commenced studying the insulator region in detail and discovered several protein binding sites, one of which, they showed, bound a protein that was necessary and sufficient for enhancer blocking. The protein, CTCF, had been known for some time to regulate gene activity; however, this new discovery suggested it might also be involved in higher order chromatin organization.
They looked for other locations where CTCF might function and found that it played a critical role in the control of the Igf2/H19 imprinted locus. This two-gene region is special in that individuals only express the paternal copy of Igf2.
Felsenfeld's group described a regulatory mechanism in which the H19 and Igf2 genes are separated by an imprinted control region containing CTCF-binding sites. The ICR on the paternal allele is methylated, preventing CTCF from binding and allowing a downstream enhancer to promote expression of both genes. The maternal allele, however, remains unmethylated and capable of binding CTCF, thus blocking enhancer activity and preventing it from driving expression of Igf2. Similar results were obtained independently in the laboratories of Shirley M. Tilghman at Princeton University and Rolf Ohlsson at the Karolinska Institutet in Sweden.
The role of CTCF now is well established, and it's been shown to function by promoting the formation of DNA loops that bring distant genetic elements physically closer. "CTCF is part of a regulatory network that's three dimensional and physical, long-range physical," Felsenfeld says enthusiastically. "We just keep going up in scale."
|A possible mechanism by which an insulator element could protect against the propagation of a silencing histone modification into a transcriptionally active chromatin domain.