A number of molecules with critical roles in chromatin organization, remodeling, and epigenetic modification have been identified in recent years. However, our understanding of which ones are the key regulators or how chromatin structure marked by a specific epigenetic modification is established is far from complete. We have presented evidence that epigenetic histone H3S10 phosphorylation by the JIL-1 kinase at interphase is a key regulator of euchromatic regions by antagonizing heterochromatization and gene silencing in Drosophila. Consequently, to understand regulation of heterochromatin formation and gene silencing in Drosophila, a premier model system for such studies, it will be crucial to determine the molecular mechanisms of the H3S10ph mark's role in this process. Based on our previous findings we propose a model where JIL-1 kinase activity and phosphorylation of histone H3S10 functions to antagonize heterochromatization by regulating a dynamic balance between factors promoting repression and activation of gene expression. The molecular mechanisms underlying this hypothesis will be explored in three specific aims: 1) In the first aim we will test the hypothesis that H3S10 phosphorylation functions to regulate the epigenetic state of euchromatin by using a LacI-tethering system to ectopically induce H3S10 phosphorylation and determine the changes in the distribution of chromatin markers that are diagnostic for active (euchromatic) or silenced (heterochromatic) chromatin. In order to test the inter-relationship of these epigenetic marks, we will furthermore determine whether combinations of targeted histone modifications can counteract or enhance changes in chromatin structure caused by the single modification. 2) In the second aim we will test the hypothesis that epigenetic H3S10 phosphorylation is sufficient to counteract heterochomatic spreading and silencing independently of gross alterations in polytene chromosome morphology. We will use PEV suppression/enhancement as a "read out" for the relative influence of H3S10 phosphorylation on gene expression. To separate out structural and catalytic contributions of JIL-1 we will express truncated and "kinase-dead" JIL-1 proteins transgenically in both wild-type and JIL-1 null mutant backgrounds and quantify the effect on PEV of different reporters. 3) In the third aim we will address the question of whether H3S10 phosphorylation is targeted to specific genomic locations. We will answer this question by specifically mapping interphase JIL-1 and H3S10 phosphorylation sites by ChIP-seq of non-dividing salivary gland chromosomes. We will use microarray analysis to identify genes whose expression levels are regulated by JIL-1-mediated H3S10 phosphorylation. We expect that the proposed studies of H3S10 phosphorylation by JIL-1 will greatly extend our knowledge of how a specific epigenetic mark modulates chromatin structure and gene regulation, a topic that is directly relevant to human development and disease including cancer.
The proposed experiments will greatly enhance our understanding of the molecular mechanisms controlling heterochromatin formation and epigenetic gene regulation by the H3S10 phosphorylation mark. Gene silencing is a critical developmental process relevant to many human health problems that include cancer.
|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|
|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|
|Girton, Jack; Wang, Chao; Johansen, Jorgen 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; 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|
|Deng, Huai; Cai, Weili; Wang, Chao et al. (2010) JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila. Genetics 185:1183-92|
|Cai, Weili; Jin, Ye; Girton, Jack et al. (2010) Preparation of Drosophila polytene chromosome squashes for antibody labeling. J Vis Exp :|
|Boeke, Joern; Regnard, Catherine; Cai, Weili et al. (2010) Phosphorylation of SU(VAR)3-9 by the chromosomal kinase JIL-1. PLoS One 5:e10042|
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