The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

In this study, we show that a reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, which is a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. We further provide evidence that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, we propose a model where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization.     

Reduced levels of Su(var)3-9 but not Su(var)2-5 (HP1) counteract the effects on chromatin structure and viability in loss-of-function mutants of the JIL-1 histone H3S10 kinase

It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase is a major regulator of chromatin structure and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of JIL-1 kinase activity, the major heterochromatin markers histone H3K9me2 and HP1 spread in tandem to ectopic locations on the chromosome arms. In this study, we show that the lethality as well as some of the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-9 gene. This effect was observed with three different alleles of Su(var)3-9, strongly suggesting it is specific to Su(var)3-9 and not to second site modifiers. This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene that codes for HP1 in Drosophila where no genetic interactions were detectable between JIL-1 and Su(var)2-5. Taken together, these findings indicate that while Su(var)3-9 histone methyltransferase activity is a major factor in the lethality and chromatin structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1.     

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

The 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. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

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

The 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. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w(m4) backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH₂-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH₂-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH₂-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.     

Loss-of-function alleles of the JIL-1 histone H3S10 kinase enhance position-effect variegation at pericentric sites in Drosophila heterochromatin

In this study we show that loss-of-function alleles of the JIL-1 histone H3S10 kinase act as enhancers of position-effect variegation at pericentric sites whereas the gain-of-function JIL-1(Su(var)3-1[3]) allele acts as a suppressor strongly supporting a functional role for JIL-1 in maintaining euchromatic chromatin and counteracting heterochromatic spreading and gene silencing.     

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.     

JIL-1 and Su(var)3-7 Interact Genetically and Counteract Each Other's Effect on Position-Effect Variegation in Drosophila

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity.SU(VAR)3-9, a histone methyltransferase, Su(var)2-5, HP1a, and Su(var)3-7, a 1250-residue zinc-finger protein are all inherent components of pericentric heterochromatin (Rea et al. 2000; Eissenberg and Elgin 2000; Schotta et al. 2002; Delattre et al. 2004; Ebert et al. 2004) and are important factors for silencing of reporter genes by heterochromatic spreading in Drosophila (for review see Weiler and Wakimoto 1995; Girton and Johansen 2008). Su(var)3-9 has been shown to catalyze most of the dimethylation of the histone H3K9 residue which in turn can promote HP1a and Su(var)3-7 recruitment (Schotta et al. 2002; Jaquet et al. 2006). In addition, Su(var)3-9, HP1a, and Su(var)3-7 can directly interact with each other, suggesting a model where interdependent interactions between Su(var)3-9, HP1a, and Su(var)3-7 lead to heterochromatin assembly at pericentric sites (Lachner et al. 2001; Schotta et al. 2002; Elgin and Grewal 2003; Jaquet et al. 2006). Heterochromatin formation in Drosophila is initiated early in development through active removal of H3K4 methylation by the LSD1 demethylase homolog Su(var)3-3 (Rudolph et al. 2007). Subsequently, a developmentally regulated balance between Su(var)3-3 H3K4 demethylase, Su(var)3-9 H3K9 methyltransferase, and RPD3 H3K9 deacetylase activity contribute to conserve the distinction between euchromatic and heterochromatic domains (Rudolph et al. 2007). Thus, highly complex interactions between multiple heterochromatic and euchromatic factors are likely to contribute to the regulation of a dynamic balance between the distinct chromatin environments promoting gene activity and gene silencing.It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase (Jin et al. 1999; Wang et al. 2001) is a major regulator of chromatin structure (Deng et al. 2005; 2008) and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing (Ebert et al. 2004; Zhang et al. 2006; Lerach et al. 2006; Bao et al. 2007). In the absence of the JIL-1 kinase, the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms with the most pronounced increase on the X chromosomes (Zhang et al. 2006; Deng et al. 2007). However, overall levels of the H3K9me2 mark and HP1a were unchanged, suggesting that the spreading was accompanied by a redistribution that reduces the levels in pericentromeric heterochromatin. Genetic interaction assays demonstrated that the lethality as well as some of the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-9 gene (Zhang et al. 2006; Deng et al. 2007). This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene where no genetic interactions were detectable between JIL-1 and Su(var)2-5 (Deng et al. 2007) Thus, these findings indicate that while Su(var)3-9 histone methyltransferase activity may be a factor in the lethality and chromatin structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1a. However, the potential role of the third major heterochromatin component, Su(var)3-7, was not addressed in these studies. Here we show that Su(var)3-7, like Su(var)3-9, genetically interacts with JIL-1, that reducing the dose of Su(var)3-7 significantly reduces the lethality of JIL-1 null mutants, and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalacing effect on position-effect variegation (PEV).     

Partitioning of the maize epigenome by the number of methyl groups on histone H3 lysines 9 and 27

We report a detailed analysis of maize chromosome structure with respect to seven histone H3 methylation states (dimethylation at lysine 4 and mono-, di-, and trimethylation at lysines 9 and 27). Three-dimensional light microscopy and the fine cytological resolution of maize pachytene chromosomes made it possible to compare the distribution of individual histone methylation events to each other and to DNA staining intensity. Major conclusions are that (1) H3K27me2 marks classical heterochromatin; (2) H3K4me2 is limited to areas between and around H3K27me2-marked chromomeres, clearly demarcating the euchromatic gene space; (3) H3K9me2 is restricted to the euchromatic gene space; (4) H3K27me3 occurs in a few (roughly seven) focused euchromatic domains; (5) centromeres and CENP-C are closely associated with H3K9me2 and H3K9me3; and (6) histone H4K20 di- and trimethylation are nearly or completely absent in maize. Each methylation state identifies different regions of the epigenome. We discuss the evolutionary lability of histone methylation profiles and draw a distinction between H3K9me2-mediated gene silencing and heterochromatin formation.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation

Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.     

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

Epigenetic indexing of chromatin domains by histone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.     

Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila

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.     

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.