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 regulation of globin gene expression as a model system. Studies are carried out to understand how globin genes are regulated during development, with emphasis on the structure and physical chemistry of the complexes between erythroid-specific regulatory factors and their target sites on DNA. The chromatin structure of the beta-globin locus and adjacent genes is being examined in great detail, in order to understand how this structure is disrupted during transcription and how boundaries are established between active and inactive chromatin domains. As part of this work, we have recently made use of high resolution methods to map the modifications of histones and of DNA that are associated with gene activation and suppression across the locus. This has revealed the involvement of certain methylated sites on histones H3 and H4 in marking transcriptionally active or inactive chromatin domains. We continue to explore these and other histone modifications and variants in order to establish the chronology of reactions that turn genes on or off. We have also examined the interplay between loss of certain histone modifications and the onset of DNA methylation during transgene silencing.

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. 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. 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. This approach appears to be a general way of studying the physical properties of macromolecular complexes present only at genomic abundance.

All of these approaches are now being extended to other loci as well in order to understand the role of chromatin boundaries in controlling gene expression. Presently attention is focused on the T cell receptor alpha and immunoglobulin heavy chain loci.

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

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

3. 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]

4. 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]

5. 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]

6. 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]

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

8. 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]

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

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

11. 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]

12. 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]

13. 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]

14. 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]

15. Prioleau MN Nony P Simpson M Felsenfeld G An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene. EMBO J (18): 4035-48, 1999. [Full Text/Abstract]

16. Bednar J Studitsky VM Grigoryev SA Felsenfeld G Woodcock CL The nature of the nucleosomal barrier to transcription: direct observation of paused intermediates by electron cryomicroscopy. Mol Cell (4): 377-86, 1999. [Full Text/Abstract]

17. 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]