The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing inDrosophila melanogaster

Abstract

SummaryThe JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein. By transgenically expressing various truncated versions of JIL-1, with or without kinase activity, and assessing their effect on PEV and heterochromatic spreading, we show that the gross perturbation of polytene chromosome morphology observed in JIL-1 null mutants is unrelated to gene silencing in PEV and is likely to occur as a result of faulty polytene chromosome alignment and/or organization, separate from epigenetic regulation of chromatin structure. Furthermore, the findings provide evidence that the epigenetic H3S10 phosphorylation mark itself is necessary for preventing the observed heterochromatic spreading independently of any structural contributions from the JIL-1 protein.Key words: JIL-1 kinase, PEV, Heterochromatin, Gene silencing, Drosophila IntroductionThe JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase (Jin et al., 1999;Wang et al., 2001). Mutational analyses have shown that the JIL-1 gene is essential for viability (Wang et al., 2001;Zhang et al., 2003) and that a reduction in JIL-1 kinase activity leads to a global disruption of polytene chromosome morphology (Wang et al., 2001;Deng et al., 2005). Furthermore, genetic interaction assays with JIL-1 hypomorphic and null allelic combinations demonstrated that the JIL-1 protein can counterbalance the effect of the three major heterochromatin components Su(var)3-9, Su(var)3-7 and Su(var)2-5 (HP1a) on position-effect variegation (PEV) (Deng et al., 2010;Wang et al., 2011). Based on these observations, it has been proposed that the epigenetic H3S10 phosphorylation mark functions to counteract heterochromatic spreading and gene silencing in Drosophila melanogaster (Ebert et al., 2004;Zhang et al., 2006;Deng et al., 2007;Deng et al., 2010). However, the previous experiments could not exclude the possibility that the effect of the JIL-1 protein on PEV was instead caused by the gross alterations of polytene chromosome morphology observed in the absence of JIL-1, or arose from structural contributions of the JIL-1 protein, independent of its H3S10 phosphorylation activity. In order to distinguish between these scenarios, we have cloned various full length and truncated versions of the JIL-1 protein into the pYES

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

“…At pericentric sites loss-of-function alleles of JIL-1 act as enhancers of PEV whereas gain-of-function alleles act as suppressors. 7 As predicted by the model, the results of Wang et al 5 showed that the level of the H3K9me2 mark at the reporter was inversely proportional to the H3S10ph level. PEV was enhanced with increased levels of H3K9me2 in the absence of H3S10 phosphorylation and PEV was suppressed with increased levels of the H3S10ph mark and a concomitant decrease in the level of the H3K9me2 mark.…”

“…[1][2][3] These observations suggested a model for a dynamic balance between euchromatin and heterochromatin, 1,[3][4][5] where the level of gene expression is determined by antagonistic functions of the euchromatic H3S10ph mark and the heterochromatic H3K9me2 mark. 3,[5][6][7][8] Wang et al 5 tested this model by transgenically expressing various truncated versions of JIL-1, with or without kinase activity and correlating their effect on PEV with the levels of the H3S10ph and H3K9me2 marks at a hsp70-white reporter gene as determined by ChIP assays in the pericentric insertion line 118E-10. At pericentric sites loss-of-function alleles of JIL-1 act as enhancers of PEV whereas gain-of-function alleles act as suppressors.…”

Epigenetic dysregulation by nickel through repressive chromatin domain disruption

“…that showed association of H3K9me2 spreading with decreased gene expression (27)(28)(29)(30). To examine this on a genome-wide scale, we stratified all of the genes into those in H3K9me2-spreading domains (3,930) and genes that are not in H3K9me2 in either control or Ni-exposed cells (8,096) ( Fig.…”

“…Ni-induced spreading of H3K9me2 is reminiscent of the local discrete changes, suggesting that misregulation of this regulatory switch results in altered gene expression. Furthermore, several earlier studies have suggested ectopic spreading of H3K9me2 to be an important signal for transcriptional silencing (27)(28)(29)(30)39). However, the alternate possibility of gene silencing resulting in spreading of H3K9me2 in a subset of the spreading domains cannot be ruled out.…”

Trans-inactivation: Repression in a wrong place

“…The JIL-1 kinase and the H3S10 histone modification counteract heterochromatin spreading. [4][5][6][7][8][9][10] According to the current model of heterochromatin formation, Su(var)3-9 HKMT methylates the lysine 9 residue of histone H3. HP1a binds to H3K9me and then recruits another molecule of Su(var)3-9 for further methylation of H3 in the adjacent nucleosome.…”