We have continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of ATP-dependent chromatin remodeling enzymes. We completed studies on the biology of ATP-dependent chromatin remodeling by Nucleosome Remodeling Factor (NURF) in the mouse. Bptf, the largest subunit of NURF, is required for maturation of thymocytes. We traced this defect to the misregulation of a number of genes important for thymocyte development and found Bptf-dependent changes in chromatin structure at gene targets as well as a requirement for NURF in the binding of the sequence specific transcription factor SRF to chromatin. This work was submitted, revised, and accepted for publication and is now published in Genes and Development. We have also completed a study on the mechanism of histone H2A.Z replacement by the SWR1 chromatin remodeling complex. We found that promoter proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae with substantial representation of nucleosomes containing one, two, or no H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes and free H2A.Z-H2B dimers leading to eviction of nucleosomal H2A-H2B and deposition of H2A.Z-H2B. These results suggest that the in vivo substrates govern the specificity and outcome of the replacement reaction. This work was submitted, revised, and accepted for publication and is now published in Cell 2010. We have continued investigations on the molecular architecture of centromere-specific nucleosomes containing histone variant CenH3. We found that DNA base composition influences reconstitution of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution is robust for non-centromere DNA, but inefficient for centromere DNA, due to an AT-rich centromere element that hinders association of Cse4-containing histone octamers. By contrast, inclusion of non-histone Scm3 with Cse4/H4 permits reconstitution on natural centromere sequences. Scm3 has a DNA binding domain that exhibits preference for the AT-rich centromere element, and a histone chaperone-like domain that promotes loading of Cse4/H4. In vivo, Scm3-GFP localizes to the centromere cluster in all phases of the cell cycle, including mitosis. Scm3 can also be cross-linked to centromeres in synchronized cells throughout the cell cycle, even when Cse4/H4 is temporarily dislodged in early S phase. These findings suggest a model in which centromere DNA-bound Scm3 aids recruitment of Cse4/H4 to assemble an H2A/H2B-deficient centromeric nucleosome. This work was submitted, revised, and accepted for publication and is now published in Molecular Cell 2011. We are now preparing two manuscripts on the mechanism of recruitment of the SWR1 chromatin remodeling enzyme and on the dynamics of the centromere specific histone variant Cse4.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Landry, Joseph W; Banerjee, Subhadra; Taylor, Barbara et al. (2011) Chromatin remodeling complex NURF regulates thymocyte maturation. Genes Dev 25:275-86
Xiao, Hua; Mizuguchi, Gaku; Wisniewski, Jan et al. (2011) Nonhistone Scm3 binds to AT-rich DNA to organize atypical centromeric nucleosome of budding yeast. Mol Cell 43:369-80
Zhou, Zheng; Feng, Hanqiao; Zhou, Bing-Rui et al. (2011) Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3. Nature 472:234-7
Li, Xingguo; Wang, Shaohua; Li, Ying et al. (2011) Chromatin boundaries require functional collaboration between the hSET1 and NURF complexes. Blood 118:1386-94
Luk, Ed; Ranjan, Anand; Fitzgerald, Peter C et al. (2010) Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome. Cell 143:725-36
Kwon, So Yeon; Xiao, Hua; Wu, Carl et al. (2009) Alternative splicing of NURF301 generates distinct NURF chromatin remodeling complexes with altered modified histone binding specificities. PLoS Genet 5:e1000574
Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas et al. (2009) N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex. J Biol Chem 284:6200-7
Kwon, So Yeon; Xiao, Hua; Glover, Bradley P et al. (2008) The nucleosome remodeling factor (NURF) regulates genes involved in Drosophila innate immunity. Dev Biol 316:538-47
Zhou, Zheng; Feng, Hanqiao; Hansen, D Flemming et al. (2008) NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B. Nat Struct Mol Biol 15:868-9