The long term objective of this study is to determine how signal transduction pathways in the nucleus regulate chromosomal architecture and gene expression. Towards this end, we have identified a novel tandem kinase in Drosophila, JIL-1, that associates with the chromosomes throughout the cell cycle, localizes specifically to the gene-active interband regions of the larval polytene chromosomes, is capable of phosphorylating histone H3 in vitro, and is enriched almost two-fold on the transcriptionally hyperactive male larval polytene X chromosome due to its association with the MSL dosage compensation complex. Histone H3 serine 10 phosphorylation levels are severely reduced in embryos from a hypomorphic JIL-1 mutant, thus placing JIL-l in the pathway mediating histone H3 phosphorylation. In addition, with high penetrance these mutants (which contain low levels of JIL-l kinase) show a number of chromosomal abnormalities, including lack of condensation at mitosis, disorganization in alignment at the mitotic spindle, separation from centrosomes, and ultimately chromosomal breakdown and fragmentation. Only 5 percent of the embryos survive to hatching, and of the animals that survive to adulthood, females outnumber males by a 2:1 ratio, supporting the hypothesis that JIL-1 is essential in dosage compensation mechanisms. Thus, these findings suggest a model where JIL-l signaling plays a direct functional role in chromosomal regulation and maintenance throughout the cell cycle, including condensation and segregation of chromosomes at metaphase as well as modification of chromatin and transcriptional regulation at interphase. We propose to test this model by generating a range of JIL-l mutant alleles, including a complete null, which will allow us to genetically dissect the functional requirements for JIL-l in different pathways and at different stages. We will determine whether histone H3 is a direct or indirect target of JIL-1 using biochemical approaches, and we will identify other potential substrates and interaction partners using genetic and yeast two-hybrid approaches. Our expectation is that JIL-l function is necessary in more than one chromatin remodeling complex, since JIL-l is found in females and on autosomes in addition to its presence on the male X-specific chromatin remodeling complex. The powerful molecular, genetic, and cellular approaches available in Drosophila are uniquely suited to the analysis of such complex signaling pathways. Thus, these studies will provide valuable new insights into the role of phosphorylation in regulation of gene expression and chromatin structure of fundamental significance for understanding both normal and cancerous developmental processes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Carter, Anthony D
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Iowa State University
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Li, Yeran; Wang, Chao; Cai, Weili et al. (2017) H2Av facilitates H3S10 phosphorylation but is not required for heat shock-induced chromatin decondensation or transcriptional elongation. Development 144:3232-3240
Wang, Chao; Li, Yeran; Cai, Weili et al. (2014) Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark. Chromosoma 123:273-80
Cai, Weili; Wang, Chao; Li, Yeran et al. (2014) Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila. Nucleic Acids Res 42:5456-67
Girton, Jack; Wang, Chao; Johansen, Jørgen et al. (2013) The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome. Fly (Austin) 7:129-33
Wang, Chao; Yao, Changfu; Li, Yeran et al. (2013) Evidence against a role for the JIL-1 kinase in H3S28 phosphorylation and 14-3-3 recruitment to active genes in Drosophila. PLoS One 8:e62484
Li, Yeran; Cai, Weili; Wang, Chao et al. (2013) Domain requirements of the JIL-1 tandem kinase for histone H3 serine 10 phosphorylation and chromatin remodeling in vivo. J Biol Chem 288:19441-9
Yao, Changfu; Ding, Yun; Cai, Weili et al. (2012) The chromodomain-containing NH(2)-terminus of Chromator interacts with histone H1 and is required for correct targeting to chromatin. Chromosoma 121:209-20
Wang, Chao; Cai, Weili; Li, Yeran et al. (2012) H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila. Fly (Austin) 6:93-7
Wang, Chao; Cai, Weili; Li, Yeran et al. (2011) The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing in Drosophila melanogaster. J Cell Sci 124:4309-17
Wang, Chao; Girton, Jack; Johansen, Jorgen et al. (2011) A balance between euchromatic (JIL-1) and heterochromatic [SU(var)2-5 and SU(var)3-9] factors regulates position-effect variegation in Drosophila. Genetics 188:745-8

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