DEVELOPMENT 229 RESEARCH ARTICLE INTRODUCTIONHigher order chromatin structure is important for epigenetic regulation and control of gene activation and silencing. In eukaryotes, a considerable proportion of the genome is packaged into constitutive heterochromatin or facultative heterochromatin that represents transiently condensed and silenced euchromatin (Schotta et al., 2003). Formation of heterochromatin and repression of transcription involves covalent modifications of histone tails and/or the exchange of histone variants (Swaminathan et al., 2005). Current evidence suggests that a major pathway in the establishment of heterochromatin is initiated by the RNAi machinery that marks prospective heterochromatic regions (Volpe et al., 2002;Pal-Bhadra et al., 2004;Verdel et al., 2004). This leads to the deacetylation of histone H3K9, followed by the dimethylation of this residue and the recruitment of HP1 (Lachner et al., 2001;Nakayama et al., 2001;Ebert et al., 2004). Thus, dimethylation of histone H3K9 and the presence of HP1 serve as major chromatin modification markers for the presence of transcriptionally silenced chromatin (Fischle et al., 2003;Swaminathan et al., 2005). However, it should be noted that HP1 recently has been demonstrated to also play a role in euchromatic gene regulation that is not linked to histone H3K9 dimethylation (Piacentini et al., 2003;Cryderman et al., 2005).Ebert et al. (Ebert et al., 2004) recently identified the Su(var)3-1 mutations as alleles of the JIL-1 locus that antagonize the expansion of heterochromatin formation in Drosophila. JIL-1 is a tandem kinase that localizes specifically to euchromatic interband regions of polytene chromosomes (Jin et al., 1999). Analysis of JIL-1 null and hypomorphic alleles showed that JIL-1 is essential for viability, and that reduced levels of JIL-1 protein lead to a global disruption of chromosome structure (Jin et al., 2000;Wang et al., 2001;Zhang et al., 2003;Deng et al., 2005). These defects are correlated with severely decreased levels of histone H3S10 phosphorylation (pH3S10), providing evidence that JIL-1 is the predominant kinase regulating the phosphorylation state of this residue at interphase (Wang et al., 2001).However, as the Su(var)3-1 alleles generate proteins with COOHterminal deletions that are dominant gain-of-function mutations, the experiments of Ebert et al. (Ebert et al., 2004) did not directly address the normal function of JIL-1. In this study, we show that the reduction in JIL-1 protein levels and histone H3S10 phosphorylation caused by hypomorphic or null loss-of-function alleles of the JIL-1 locus results in the spreading of the major heterochromatin markers dimethyl H3K9 (dmH3K9) and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Furthermore, genetic interaction assays demonstrated that JIL-1 functions antagonistically to Su(var)3-9, which is the major catalyst for dimethylation of the histone H3K9 residue (Schotta et al., 2002). These findings suggest a mode...

Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6 and dKeap1EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.

The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions.

In this study we have generated two new hypomorphic Chro alleles and analyzed the consequences of reduced Chromator protein function on polytene chromosome structure. We show that in Chro 71 /Chro 612 mutants the polytene chromosome arms were coiled and compacted with a disruption and misalignment of band and interband regions and with numerous ectopic contacts connecting non-homologous regions. Furthermore, we demonstrate that Chromator co-localizes with the JIL-1 kinase at polytene interband regions and that the two proteins interact within the same protein complex. That both proteins are necessary and may function together is supported by the finding that a concomitant reduction in JIL-1 and Chromator function synergistically reduces viability during development. Overlay assays and deletion construct analysis suggested that the interaction between JIL-1 and Chromator is direct and that it is mediated by sequences in the C-terminal domain of Chromator and by the acidic region within the C-terminal domain of JIL-1. Taken together these findings indicate that Chromator and JIL-1 interact in an interband-specific complex that functions to establish or maintain polytene chromosome structure in Drosophila.

ser2), indicating that Pol IIo ser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. This is supported by the finding that there is no change in the levels of Pol IIo ser2 in JIL-1 null mutant backgrounds compared with wild type. mRNA from the six genes that encode the major heat shock protein in Drosophila, Hsp70, is transcribed at robust levels in JIL-1 null mutants, as directly demonstrated by qRT-PCR. Taken together, these data are inconsistent with the model that Pol II-dependent transcription at active loci requires JIL-1-mediated histone H3S10 phosphorylation, and instead support a model in which transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin.

*Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a 'kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.KEY WORDS: Histone H3S10 phosphorylation, Chromatin structure remodeling, JIL-1 kinase, Drosophila Development 135, 699-705 (2008)

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

High-density genetic maps (HDGMs) are very useful for genomic studies and quantitative trait loci (QTL) mapping. However, the low frequency of DNA polymorphisms in peanut has limited the quantity of available markers and hindered the construction of a HDGM. This study generated a peanut genetic map with the highest number of high-quality SNPs based on specific locus amplified fragment sequencing (SLAF-seq) technology and a newly constructed RIL population (“ZH16” × “sd-H1”). The constructed HDGM included 3,630 SNP markers belonging to 2,636 bins on 20 linkage groups (LGs), and it covers 2,098.14 cM in length, with an average marker distance of 0.58 cM. This HDGM was applied for the following collinear comparison, scaffold anchoring and analysis of genomic characterization including recombination rates and segregation distortion in peanut. For QTL mapping of investigated 14 yield-related traits, a total of 62 QTLs were detected on 12 chromosomes across 3 environments, and the co-localization of QTLs was observed for these traits which were significantly correlated on phenotype. Two stable co-located QTLs for seed- and pod-related traits were significantly identified in the chromosomal end of B06 and B07, respectively. The construction of HDGM and QTL analysis for yield-related traits in this study provide useful information for fine mapping and functional analysis of genes as well as molecular marker-assisted breeding.