The National Institute of Diabetes & Digestive & Kidney Diseases

Our group studies the relationship between chromatin structure and gene expression in eukaryotes, with particular interest in epigenetic mechanisms and the structure of both the individual nucleosomes (the fundamental chromatin subunits) and the folded polynucleosome fiber. We have investigated recently the role of histone variants in regulation of chromatin structure and gene expression.  We have shown that the variants H3.3 and H2A.Z,  when present together in the same nucleosome core particle (NCP), result in much less stable NCPs than those containing the most abundant variants, H3.1 and H2A.   We have determined the genome-wide distribution of these unstable NCPs and find that they are concentrated at the promoters of transcriptionally active genes, as well as at sites containing transcriptional regulatory elements.  These results support a model in which such unstable NCPs play a role in making active promoters more accessible for binding by regulatory factors.    

We also study long range chromatin organization and the boundaries between independently regulated domains, which play a role in regulation of gene expression. Here we have focused on the properties of insulator elements that help to establish such boundaries. We have identified two kinds of boundary function, one that blocks inappropriate activation of a promoter by a distal enhancer, and a second that acts as a barrier against encroachment of heterochromatin into adjacent open chromatin regions. In each case we have identified proteins that bind to the insulator sites, as well as co-factors which those proteins recruit, and in each case the result suggests how the enhancer blocking or barrier activity arises; present work is examining these mechanisms in detail, both by biochemical and functional analysis of the complexes, and by studies of the effects of protein knockdown on the local and long range chromatin structure and histone modification patterns. We are presently investigating the mode of action of one such protein (VEZF1/BGP1) that blocks DNA methylation, a silencing signal, from modifying a protected reporter gene.  We have shown that cells lacking this protein have widespread changes in their DNA methylation patterns, which appears to be an indirect effect of VEZF1 action.  Another area of interest concerns the properties of the extended condensed chromatin region, upstream of the beta globin locus, which appears to be a model heterochromatic structure. We have excised this region as a homogeneous chromatin fragment containing about 16 kb of DNA, and studied its hydrodynamic properties in a highly precise fashion using a combination of preparative and analytical ultracentrifugation, taking advantage of our strong experience with solution physicochemical measurements of macromolecules. We are now investigating the biochemical mechanisms that establish and maintain the heterochromatic state of this fragment.

All of these approaches are now being extended to other loci.  In particular we are studying long range interactions within the nucleus in human pancreatic beta cells, between the insulin gene and other genes that may be co-regulated.  This is part of a larger effort to study chromatin structure of the insulin locus and its relationship to insulin gene expression and secretion.

1. Jin, C., Zang, C., Wei, G.,  Cui, K. , Peng, W.,  Zhao, K.  and Felsenfeld, G. H3.3/H2A.Z Double variant-containing nucleosomes mark ‘nucleosome-free regions’ of active promoters and other regulatory regions in the human genome.    Nature Genetics 41:941-945  (2009).

2. Mutskov, M. and Felsenfeld, G.    The human insulin gene is part of a large open chromatin domain specific for human islets.   Proceedings of the National Academy of Sciences USA, in press (2009)

3. Ghirlando, R. and Felsenfeld, G. Hydrodynamic studies on defined heterochromatin fragments support a 30 nm fiber having 6 nucleosomes per turn  Journal of Molecular Biology, 376:1417-25 (2008)

 4. Gowher, H. and Felsenfeld, G.  Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b.   Genes & Development, 22:2075-2084 (2008)

5. Jin, C. and Felsenfeld, G. Nucleosome stability mediated by histone variants H3.3 and H2A.Z.  Genes & Development 21:1519-1529 (2007).

6. Jin C, Felsenfeld G Proc Natl Acad Sci U S A (103): 574-9, 2006. [Full Text/Abstract]

7. Huang S, Litt M, Felsenfeld GGenes Dev (19): 1885-93, 2005. [Full Text/Abstract]

8. Felsenfeld G, Burgess-Beusse B, Farrell C, Gaszner M, Ghirlando R, Huang S, Jin C, Litt M, Magdinier F, Mutskov V, Nakatani Y, Tagami H, West A, Yusufzai TCold Spring Harb Symp Quant Biol (69): 245-50, 2004. [Full Text/Abstract]

9. Studitsky VM Walter W Kireeva M Kashlev M Felsenfeld G Chromatin remodeling by RNA polymerases. Trends Biochem Sci (29): 127-35, 2004. [Full Text/Abstract]

10. Yusufzai TM Tagami H Nakatani Y Felsenfeld G CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell (13): 291-8, 2004. [Full Text/Abstract]

11. Ghirlando R Litt MD Prioleau MN Recillas-Targa F Felsenfeld G Physical properties of a genomic condensed chromatin fragment. J Mol Biol (336): 597-605, 2004. [Full Text/Abstract]

12. West AG, Huang S, Gaszner M, Litt MD, Felsenfeld GMol Cell (16): 453-63, 2004. [Full Text/Abstract]

13. Mutskov V Felsenfeld G Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J (23): 138-49, 2004. [Full Text/Abstract]

14. Felsenfeld G Groudine M Controlling the double helix. Nature (421): 448-53, 2003. [Full Text/Abstract]

15. West AG Gaszner M Felsenfeld G Insulators: many functions, many mechanisms. Genes Dev (16): 271-88, 2002. [Full Text/Abstract]

16. Mutskov VJ Farrell CM Wade PA Wolffe AP Felsenfeld G The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation. Genes Dev (16): 1540-54, 2002. [Full Text/Abstract]

17. Litt MD Simpson M Gaszner M Allis CD Felsenfeld G Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science (293): 2453-5, 2001. [Full Text/Abstract]

18. Litt MD Simpson M Recillas-Targa F Prioleau MN Felsenfeld G Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J (20): 2224-35, 2001. [Full Text/Abstract]

19. Bell AC Felsenfeld G Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature (405): 482-5, 2000. [Full Text/Abstract]

20. Bell AC West AG Felsenfeld G The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell (98): 387-96, 1999. [Full Text/Abstract]