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MAPPING HIGHER ORDER CHROMATIN STRUCTURES
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Bruce H. Howard, MD, Head, Human Genetics Section |
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| Our research focuses on the role of higher order chromatin structure in regulating gene expression. A topic of special interest is epigenetic memory, both how it is established and what determinants influence its stability. Our central hypothesis holds that errors in epigenetic memory contribute to developmental defects, cancer, and age-related diseases. Techniques under development assess the type and extent of chromatin remodeling that accompanies developmental programs, terminal differentiation, or senescence. In associated work, we perform structure-function studies to understand the roles played by specific chromatin regulatory gene products in establishing or maintaining epigenetic memory. Genome sampling, bioinformatics, and high-throughput approaches to map higher order chromatin domains Russanova, Howard Several examples in the literature provide direct or indirect evidence for chromatin remodeling in conjunction with differentiation, cancer, or aging. Most such examples derive from serendipitous observations; thus, we have only a limited understanding of the extent and types of chromatin changes. Differential display is well established as a systematic means to screen for alterations in mRNA levels. We have combined chromatin immunoprecipitation (ChIP) with a modified form of differential display in order to screen for regions of unusual chromatin structure or domains of remodeling. In most genome sampling experiments to date, chromatin fragments in the ChIP step were fractionated according to histone acetylation level. Elevated levels of acetylation are typically associated with active gene transcription, whereas underacetylation correlates with repression or silencing. The human genome is organized into alternating gene-rich and gene-poor regions. An important validation of the ChIP genome sampling approach is provided by the observation that extremely underacetylated loci identified in the screens uniformly reside within gene-poor regions. By contrast, highly acetylated loci are found to lie predominantly within gene-rich regions. These differences are highly significant and would not be observed if chromatin fragment immunoprecipitation yielded loci at random with respect to higher order chromatin domains. The HL-60 promyelocytic cell line has served as a useful model system in which to search for domains of chromatin remodeling. HL-60 cells differentiate into macrophage-like cells in response to treatment with phorbol esters. A screen of several thousand revealed several loci in which terminal differentiation along the macrophage lineage is accompanied by elevated histone acetylation. One such locus, located on chromosome 2 at a position 40 kb upstream from the EML4 gene locus, exhibits on average a six-fold increase in acetylation. Searches for chromatin remodeling in the contexts of development and aging will require considerably improved approaches for mapping chromatin domains. To this end, we have developed an integrated system for high-throughput primer design, PCR product analysis, and graphic display of results. Novel regions of chromatin structure are likely initiated by combinations of sequence-specific DNA binding factors. To seed the mapping of such regions, we have developed a set of custom search algorithms that allow comparisons of mouse and human genomes for the presence of conserved binding sites. In excess of 100,000 sites per genome can be analyzed, followed by more than 10 billion blast comparisons, to yield potential binding sites of interest. The sites can be prioritized according to proximity to CpG islands, genes, and so forth. The in silico predictions can then be rapidly tested by chromatin immunoprecipitation experiments coupled with the high-throughput approaches noted above. Humphrey GW, Wang Y, Russanova VR, Hirai T, Qin J, Nakatani Y, Howard BH. Stable histone deacetylase complexes distinguished by the presence of SANT domain proteins CoREST/ kiaa0071 and Mta-L1. J Biol Chem 2001;276:6817-6824. Histone H3 methylation marks the murine H19-Igf2-imprinted region Tchernov; in collaboration with Pfeifer, Stewart The murine H19-Igf2-imprinted region is characterized by maternal expression of the H19 locus versus paternal expression of the Igf2 gene. A differentially methylated region (DMR) is located upstream from the H19 locus. In maternal cells, the DMR is unmethylated, binds to the transcription factor CTCF, and functions as an insulator to prevent activation of the Igf2 promoter by enhancers located downstream from H19. Conversely, in paternal cells, the DMR is methylated and, with respect to transcriptional and insulator activities, repressed. Histone H3 methylation at lysine position 9 (K9) appears to precede DNA methylation as a mark for transcriptional silencing in a number of systems. Experiments undertaken with collaborators revealed a high level of histone H3-K9 methylation near the H19-associated DMR and promoter region in paternal but not maternal cells. By contrast, H3-K9 methylation was detected at control regions for the Igf2 locus in paternal cells only. Of note, bisulfite mapping of DNA CpG-methylation sites confirmed that H3-K9 and DNA methylation occur with opposite parental bias upstream from the Igf2 gene. Moreover, the differential H3-K9 methylation defined an upstream region distinct from that marked by DNA methylation. This work reveals considerably greater complexity in the relative histone and DNA methylation patterns than previously appreciated. Histone deacetylase and acetylase complexes Bae; in collaboration with Ozato As noted above, histone deacetylation is typically associated with transcriptional silencing. Numerous transcriptional repressors recruit RPD3-family deacetylases. Previous work by this group and others revealed that members of this family, including HDAC1, HDAC2, and HDAC3, function within multiprotein complexes to mediate repression. Complexes including the transcription factor co-REST together with HDAC1 or HDAC2, first delineated by this group, have emerged as highly important in nervous system development. The NAD-dependent deacetylase SIR2 is essential for silencing in budding yeast, and SIR2-like proteins comprise a highly conserved family ranging from yeast to mammals. Much remains to be learned as to whether Sirtuins, the mammalian SIR2 homologs, function in transcriptional regulation. Our experiments reveal that Sirtuin2 interacts with the homeodomain protein Hox-a10. We demonstrated interaction in the yeast two-hybrid system with in vitro-translated products and by coimmunoprecipitation from mammalian cell extracts. Overexpression of Sirtuin2 counteracts transcriptional activation by Hox-a10. The results establish a link between mammalian SIR2 homologs and transcriptional control; given that Hox-a10 plays a role in genito-urinary tract formation, it is possible that Sirtuin2 might modulate the establishment or maintenance of repressive chromatin domains during development. Yamagoe S, Kanno T, Kanno Y, Sasaki S, Siegel RM, Lenardo MJ, Humphrey G, Wang Y, Nakatani Y, Howard BH, Ozato K. Interaction of histone acetylases and deacetylases in vivo. Mol Cell Biol 2003;23:1025-1033.
COLLABORATORS Keiko Ozato, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda MD Karl Pfeifer, PhD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda MD Colin Stewart, PhD, Cancer and Developmental Biology Laboratory, NCI, Frederick MD For further information, contact howardbr@mail.nih.gov |
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