The overall goal of this project is to understand the in vivo function of histones and their role in chromatin differentiation. These studies are greatly facilitated by the remarkable nuclear dimorphism of Tetrahymena in which transcription occurs specifically in vegetative somatic macronuclei and mitosis and meiosis occurs only in germline micronuclei, We are performing in vivo analyses of histone function using methods we developed for mass transformation, which occurs by homologous integration, facilitating gene knockout and gone replacement in the germline micronucleus, the somatic macronucleus, or both. T. thermophila may be second only to Saccharomyces cerevisiae in the depth to which its histone primary sequence variants and secondary modification sites have been characterized and the degree to which these have been associated with nuclear processes in different physiological and developmental states. Importantly, some of the features of the Tetrahymena histone complement and its modifications resemble those of mammals that are lacking in S. cerevisiae. Assisted by the newly available sequence of the Tetrahymena (macronuclear) genome, we have identified all of the genes that encode histones. These include three new genes whose function we propose to study: novel, highly divergent H2A, a likely H3 replacement variant and a likely centromereic H3 homologous to Cenp-A. We will continue studies on the function of phosphorylation of linker histones, proposing to test our hypothesis that it regulates gone expression by site-specific de-phosphorylation in chromatin. We will test the hypothesis that H2A.Z has a general function to limit the spread of telomeric heterochromatin. We will analyze the functions of phosphorylation and ubiquitination of H2A and H2B, determining whether these modifications have redundant or unique functions. We will test the hypothesis that phosphorylation of specific residues flanking the H2A ubiquitinafion site serves as a binary switch to regulate ubiquitination. We will use our large array of Tetrahymena strains expressing site-specific mutations of histone modification sites, coupled with TAP tagging, to identify proteins that recognize these modifications. Finally, using mutations in genes encoding molecules that function in the RNAi machinery required for heterochromatin formation, we will test the hypothesis that the centromeric H3 is targeted to centromere DNA sequences by an RNAi-like mechanism. Given the conservation of histones and their secondary modifications, and their demonstrated importance in disease, we expect the studies proposed here to provide important insights into the functions of linker histones, histone variants and histone modification in transcription and chromosome function in eukaryotes. ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Carter, Anthony D
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University of Rochester
Schools of Arts and Sciences
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Song, Xiaoyuan; Bowen, Josephine; Miao, Wei et al. (2012) The nonhistone, N-terminal tail of an essential, chimeric H2A variant regulates mitotic H3-S10 dephosphorylation. Genes Dev 26:615-29
Xiong, Jie; Lu, XingYi; Lu, YuMing et al. (2011) Tetrahymena Gene Expression Database (TGED): a resource of microarray data and co-expression analyses for Tetrahymena. Sci China Life Sci 54:65-7
Noto, Tomoko; Kurth, Henriette M; Kataoka, Kensuke et al. (2010) The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell 140:692-703
Xiong, Jie; Feng, Lifang; Yuan, Dongxia et al. (2010) Genome-wide identification and evolution of ATP-binding cassette transporters in the ciliate Tetrahymena thermophila: A case of functional divergence in a multigene family. BMC Evol Biol 10:330
Wang, Zhe; Cui, Bowen; Gorovsky, Martin A (2009) Histone H2B ubiquitylation is not required for histone H3 methylation at lysine 4 in tetrahymena. J Biol Chem 284:34870-9
Fu, Chengjie; Xiong, Jie; Miao, Wei (2009) Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila. BMC Genomics 10:208
Miao, Wei; Xiong, Jie; Bowen, Josephine et al. (2009) Microarray analyses of gene expression during the Tetrahymena thermophila life cycle. PLoS One 4:e4429
Bednenko, Janna; Noto, Tomoko; DeSouza, Leroi V et al. (2009) Two GW repeat proteins interact with Tetrahymena thermophila argonaute and promote genome rearrangement. Mol Cell Biol 29:5020-30
Aronica, Lucia; Bednenko, Janna; Noto, Tomoko et al. (2008) Study of an RNA helicase implicates small RNA-noncoding RNA interactions in programmed DNA elimination in Tetrahymena. Genes Dev 22:2228-41
Song, Xiaoyuan; Gorovsky, Martin A (2007) Unphosphorylated H1 is enriched in a specific region of the promoter when CDC2 is down-regulated during starvation. Mol Cell Biol 27:1925-33

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