Transcriptional Repression of Hox Genes by C. elegans HP1/HPL and H1/HIS-24

Elucidation of the biological role of linker histone (H1) and heterochromatin protein 1 (HP1) in mammals has been difficult owing to the existence of a least 11 distinct H1 and three HP1 subtypes in mice. Caenorhabditis elegans possesses two HP1 homologues (HPL-1 and HPL-2) and eight H1 variants. Remarkably, one of eight H1 variants, HIS-24, is important for C. elegans development. Therefore we decided to analyse in parallel the transcriptional profiles of HIS-24, HPL-1/-2 deficient animals, and their phenotype, since hpl-1, hpl-2, and his-24 deficient nematodes are viable. Global transcriptional analysis of the double and triple mutants revealed that HPL proteins and HIS-24 play gene-specific roles, rather than a general repressive function. We showed that HIS-24 acts synergistically with HPL to allow normal reproduction, somatic gonad development, and vulval cell fate decision. Furthermore, the hpl-2; his-24 double mutant animals displayed abnormal development of the male tail and ectopic expression of C. elegans HOM-C/Hox genes (egl-5 and mab-5), which are involved in the developmental patterning of male mating structures. We found that HPL-2 and the methylated form of HIS-24 specifically interact with the histone H3 K27 region in the trimethylated state, and HIS-24 associates with the egl-5 and mab-5 genes. Our results establish the interplay between HPL-1/-2 and HIS-24 proteins in the regulation of positional identity in C. elegans males.


Introduction
Linker histone H1 and heterochromatin protein HP1 are involved in numerous processes ranging from stabilizing heterochromatin condensation to the regulation of gene expression [1][2][3][4][5]. As has been reported, a methylation mark on vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of HP1 binding to linker histone has not been determined [6].
The functions of HP1 and H1 proteins are mainly dependent on the cell type in which particular variants are expressed. Although the number of H1 (11) and HP1 variants (3) presents difficulties in studying the effect of H1 and HP1 depletion in mice, some data has emerged [3,[7][8][9][10]. For example, loss of HP1b results in defective development of neuromuscular junctions and the cerebral cortex [10], whereas depletion of three of eleven H1 genes causes lethality connected with a very broad range of defects in mice [11][12]. In ES cells, the lack of three somatic H1 variants leads to changes in nucleosome spacing and local chromatin compaction, and this is correlated with decreased levels of H3K27 trimethylation [11]. Additionally, H1 is necessary to establish and maintain the DNA methylation pattern in a subset of genes including the reproductive homeobox (Rhox) gene cluster [13].
C. elegans possesses eight linker histone variants and two HP1 homologues, HPL-1 and HPL-2 [14][15][16]. Mutation of hpl-2 results in defective vulval and germline development at elevated temperatures [15][16][17]. hpl-1, in contrast to hpl-2, does not have visible effects on C. elegans development at different temperatures, however, hpl-1 acts redundantly with hpl-2 to control larval development, somatic gonad development and vulval cell fate determination [17]. Our previous study revealed that HPL-1 recognizes the linker histone variant HIS-24 when it is monomethylated at lysine 14 (HIS-24K14me1), similar to the situation in vertebrates [16]. Additionally, we showed that HIS-24 interacts with H3K27me3 [18]. The H3K27me3 modification correlates with a repressive chromatin state that inhibits expression of many developmentally regulated genes. This is consistent with studies of Hox loci demonstrating that enrichment of H3K27me3 recruits the binding of Polycomb group proteins (PcG) [19].
The Hox genes encode conserved homeodomain-containing transcription factors that control the positional identities of cells along the anterior-posterior axis [20][21]. The expression pattern of Hox genes appears to be regulated by two evolutionarily conserved PcG complexes, the ESC/E(Z) complex and the PRC1 complex. Both have been identified in flies and mammals and are linked to modulation of repressive chromatin structures [21]. The C. elegans Hox cluster consisting of lin-39, ceh-13, mab-5 and egl-5 (orthologs of Drosophila Scr, labial, ftz and Abd-B, respectively) is quite degenerated in comparison to Hox clusters in other species [22] but, as in mammals, is also globally repressed by Polycomb group (PcG) proteins [20,23]. Mutations in mes-2 and mes-6, which encode the C. elegans ESC/E(Z) complex, result in ectopic expression of Hox genes [23]. A similar phenotype has also been observed in the absence of sop-2 or sor-1 genes. SOP-2 and SOR-1 form another C. elegans PcG-like complex which shares many structural and functional properties with the Drosophila PRC1, and is involved in the global repression of Hox gene expression. Loss of sop-2 and sor-1 results in gross homeotic transformations [24][25].
To elucidate the function of H1 and HP1 related proteins in C. elegans, we decided to generate double and triple mutants, since hpl-1, hpl-2 and his-24 deficient nematodes are viable, and since HIS-24K14me1 is recognized by HPL-1 [16][17]26]. We performed global transcriptional analyses of single, double and triple mutant animals, and we found that HPL-1/-2 and HIS-24 regulate a relatively small number of genes. We provide evidence that the methylated form of HIS-24 (HIS-24K14me1) and HPL-2 are involved in the regulation of mab-5 and egl-5 expression by binding to H3K27me3, although HIS-24K14me1 does not interact with HPL-2 [16]. Furthermore, we observed that HIS-24 and HPL-2 act in parallel pathway as MES (PcG) proteins, and loss of their activity causes defects of male tail structures. Overall, our data suggest a common and dual role for C. elegans H1 and HP1, functioning both as chromatin architectural proteins and at the same time as modifiers of a small subset of genes. Furthermore, we provide the first direct evidence for redundant functions of H1 and HP1 in metazoan development.

Results
HP1 and HIS-24 are not global repressors of transcriptional activity in C. elegans C. elegans contains two related HP1 proteins (HPL) and eight linker histone variants [14][15]. Only one of the eight linker histone variants, HIS-24 is important for germline development, with its absence resulting in reduced fertility and de-repression of extrachromosomal transgenic arrays in the germline [14]. As we previously reported, the absence of HIS-24 did not affect protein levels of the other histone variants, in contrast to the mammalian H1 subtypes which are sufficient to compensate for the loss of a single linker histone [7,16]. Furthermore, we showed that C. elegans heterochromatin protein 1 variant, HPL-1 recognizes and binds the methylated form of HIS-24 [16]. Given the physical interaction of HPL-1 with HIS-24 mono-methylated at lysine 14 and their role in chromatin silencing and germline developmental processes [15][16][17], we decided to study HPL and HIS-24 function in transcriptional regulation in C. elegans. It was of great interest to determine how the HPL subtypes (HPL-1 and HPL-2) and HIS-24 affect gene expression. To determine the contribution of HIS-24 and HPL-1/-2 to the control of gene transcription, we compared the gene-expression profiles of single null mutations in the hpl-1, his-24 and hpl-2 as well as profiles of hpl-1his-24, and hpl-2; his-24 double, and hpl-2; hpl-1his-24 triple mutant animals in L4 larval stages grown at 21uC. We decided to use L4 larval stages because HIS-24 is the most abundant linker histone H1 variant at this stage according to mass spectrometry-based protein expression data ( Figure 1).
By microarray we observed very few changes in the gene expression profiles of either single, double, or triple mutants when compared with wild type animals at L4 larval stages. Among the 16,278 target probe sets assayed, we identified only modest changes in expression of just a small number of genes (Figure 2A-2H, Table 1). The majority of genes exhibiting changes were upregulated (6.5%) in the absence of the three heterochromatin components HIS-24, HPL-1 and HPL-2, in contrast to 3.7% downregulated genes from a total of 16,278 genes analyzed (FDR,0.05) suggesting that HPL-1/-2 and HIS-24 are not global repressors of transcriptional activity ( Table 1). The deletion of both hpl-1 and hpl-2 genes caused up-regulation of 4.5% genes and downregulation of 2.1% of a total 16,278 genes when compared to wild type (WT) animals.
In conclusion, deletion of the different HPL variants and HIS-24 caused an alteration in the expression of a limited number of genes, different in each HPL variant and HIS-24. Most of the genes are affected by a single HPL variant and HIS-24, supporting the theory that HPL isoforms or HIS-24 play specific roles in gene expression. Nonetheless, a proportion of genes are altered by more

Author Summary
Linker histone (H1) and heterochromatin protein 1 (HP1) play central roles in the formation of higher-order chromatin structure and gene expression. Recent studies have shown a physical interaction between H1 and HP1; however, the biological role of histone H1 and HP1 is not well understood. Additionally, the function of HP1 and H1 isoform interactions in any organism has not been addressed, mostly due to the lack of knockout alleles. Here, we investigate the role of HP1 and H1 in development using the nematode C. elegans as a model system. We focus on the underlying molecular mechanisms of gene co-regulation by H1 and HP1. We show that the loss of both HP1 and H1 alters the expression of a small subset of genes. C. elegans HP1 and H1 have an overlapping function in the same or parallel pathways where they regulate a shared target, the Hox genes.
than one HPL variant as well as HIS-24, suggesting redundant roles for HIS-24 and HPL variants, and for HPL-1/-2 may also exist.
HIS-24 acts synergistically with HPL proteins to allow normal reproduction, somatic gonad development, and vulval cell fate decisions In parallel to microarray analysis we investigated the biological role of HIS-24 and HPL proteins in C. elegans. For morphological defects we scored hpl-1(tm1624) his-24(ok1024), and hpl-2(tm1489); his-24(ok1024) double mutants as well as hpl-2(tm1489); hpl-1(tm1624) his-24(ok1024) triple mutant animals. In particular, we focused on germline nuclei morphology, hermaphrodite vulval development and the somatic patterning of the male tail since these tissues are known to be affected by mutations in chromatin factors, and HPL-2 influences vulval cell fate specification in the synMuv (synthetic multivulva) pathway [14][15]27]. We found that the deletion of hpl-2(tm1489) together with his-24(ok1024) results in synergistic non-lethal defects of vulval cell fate specification (everted vulva, multivulva) and sterility at 21uC, and at 25uC ( Table 2). While the observed phenotypic effects at 21uC were minor in contrast to the situation at 25uC, it is tempting to speculate that the effects can be also modulated through unknown mechanisms, environmental cues (temperature), which in itself may also lead to significant side-effects. Additionally, decreased brood sizes were observed in hpl-2(tm1489); his-24(ok1024) double and hpl-2(tm1489); hpl-1(tm1624) his-24(ok1024) triple mutant   (Figure 3). The brood size of the hpl-2(tm1489); his-24(ok1024) was strongly decreased by 35% of wild type worms, and was further decreased to about 50% in the hpl-2(tm1489); hpl-1(tm1624) his-24(ok1024) triple mutant animals ( Figure 3). These results were consistent with our microarray data analysis that revealed differential expression of genes involved in the embryonic development or reproduction (Table S1). Furthermore, we observed several defects in the morphology of the somatic gonad of hpl-1(tm1624) his-24(ok1024) double mutant animals grown at 21uC. In wild type, single mutant and hpl-2(tm1489); his-24(ok1024) double mutant the gonad arms form an U-shaped structure ( Figure 4A-4D). In contrast, in the double mutant hpl-1(tm1624) his-24(ok1024) 25% of gonad arms (161 of 642) form a loop ( Figure 4E). These results suggest that both proteins HIS-24 and HPL-1 are involved in the somatic gonad development whereas HIS-24 and HPL-2 influence vulva cell fate specification and reproduction ( Table 2).

HIS-24 and HPL-2 are required for chromatin compaction
Since HPL-2 and HIS-24 are required for germline development and for the chromatin based germline-specific silencing mechanism [14][15]26], we asked whether they influence the structure of nuclei. In-depth analysis revealed that the germline nuclei of hpl-2(tm1489); his-24(ok1024) double mutants differ in size and morphology when compared to single mutants or to wild type worms grown at 21uC ( Figure 4F-4I, 4L). The observed chromatin of 86% of gonad arms (36 of 42) had a more open, relaxed structure suggesting that HIS-24 and HPL-2 play a function in chromatin condensation in the germline ( Figure 4J, 4M). To assess the specific requirements for HIS-24 among the H1 isoforms, we also tested hpl-2(tm1489);hil-3(ok1556) double mutant strain to determine if the observed changes in the chromatin compaction is linker histone variant specific ( Figure 4K). As shown, loss of hpl-2 and linker histone variant hil-3 did not cause defects in chromatin compaction in contrast to hpl-2; his-24 strain. In addition, we also did not observe involvement of HPL-2 and HIL-3 on brood size ( Figure 3).
To determine if the loss of HIS-24 and HPL proteins also influence chromatin histone modifications as well as core histone H3 level, we performed western blot analysis of mutant animals. No gross changes were observed in the methylation and core histone H3 levels using antibodies directed against H3K9me3, H3K27me3, and H3 ( Figure S1). In addition, we did not detect changes in chromatin modification marks on a cellular level by immunofluorescence (data not shown) indicating that the observed effects of chromatin compaction are not correlated with alterations of histone modifications in hpl-2(tm1489); his-24(ok1024) double mutant animals.
The wild type male tail possesses nine pairs of bilateral sensory rays that function in locating and mating with hermaphrodites. Normally, the posterior hypodermal blast cells V5 and V6 produce six pairs of rays (ray 1 to ray 6), while the blast cell T gives rise to the three rays (rays 7-9) [23][24][25]. We found that mutations in both his-24 and hpl-2 (37%, 51 of 73 males with defected rays) as well as in his-24, hpl-1 and hpl-2 (83%, 76 of 107 males with defected rays) cause abnormalities in patterning of blast cells V that result in fused and atypical (under-developed) rays, while the single and hpl-1; hpl-2 and hpl-1 his-24 double mutations have normal development of rays (Table S2, Figure 5A-5E, 5G). Although hpl-1 mutation alone or in combination with his-24 or hpl-2 had no visible effect on the male tail at 21uC ( Figure 5C, 5G-5H), it appeared to be partially redundant in combination with hpl-2 and his-24 double mutations. As Figure 5J and Table S2 show, the number of under-developed rays is significantly increased (up to 42%, 39 of 107 males) in the hpl-2(tm1489); hpl-1(tm1624) his-24(ok1024) triple mutant compared to the hpl-2(tm1489); his-24(ok1024) double mutant males (13%, 18 of 73 males) ( Figure 5I). This synergism suggests that hpl-1 only in combination with his-24 and hpl-2 plays functions in the patterning of the male tail. We also tested hil-3; hpl-2 double mutant animals for the mail tail phenotype. We did not observe any defects in the patterning of the male tail of hil-3; hpl-2 double mutant animals in contrast to hpl-2; his-24 animals suggesting that HIS-24 (in combination with HPL-2) specifically affects the patterning of the mating structures in C. elegans ( Figure 5F-5I). genes in C. elegans. The molecular functions and the percentage of the total for each group are indicated. HIS-24 and HPL-2 are required for inhibiting the ectopic expression of mab-5 and egl-5 Hox genes In agreement with previous observations we analyzed the ability of his-24, hpl-1 and hpl-2 genes to regulate mab-5 and egl-5 expression [28][29]. Interestingly, these two Hox genes are required for V ray development [23] and mab-5 was slightly upregulated in our microarray analysis of hpl-2(tm1489); hpl-1(tm1624) his-24(ok1024) mutant animals (data not shown).
To verify HPL-1 depletion directly and to examine the extent of HPL-1 knockdown we tested the hpl-1 depleted hpl-2; his-24 mutant animals for presence of HPL-1 on the western blot. We found that hpl-1RNAi strongly reduces HPL-1 level compared to the controls ( Figure S2).
Since mutations in hpl-2 and his-24 affect transgene expression in C. elegans [14][15] we assessed the expression level of the endogenous EGL-5 in hpl-2; his-24 double mutant males. Western blot of hpl-2; his-24 double mutant males probed with EGL-5 antibody revealed an increased level of endogenous EGL-5 protein of predicted size (26 kDa) compared to EGL-5 level of wild type C. elegans and egl-5::gfp transgenic line ( Figure 6F) [29]. Altogether, these results suggest that HIS-24 and HPL-2 silence the Hox gene cluster, either by general repression of the transcriptional activity, or through a specific biochemical and structural function in Hox gene silencing.  HIS-24 binds to egl-5 and mab-5 loci Since HIS-24 and HPL-2 are required for inhibiting the ectopic expression of mab-5 and egl-5 Hox genes, we tested if HIS-24 and HPL-2 bind directly to their promoters in vivo and therefore regulate egl-5 and mab-5 transcription. The primer sets used for quantitative ChIP-PCR (qChIP-PCR) analysis were directed to the promoters, introns and 39UTR regions of mab-5 and egl-5 genes. Remarkably, mab-5 and egl-5 are tightly clustered on chromosome III, suggesting that chromatin structure coordinately regulates the expression of these genes ( Figure 7A). qChIP-PCR analysis revealed that HIS-24 is indeed associated with the promoters and introns of mab-5 and egl-5 genes ( Figure 7B). In contrast, we did not see any HIS-24 binding to 39UTR regions ( Figure 7B). However they are occupied by H3 ( Figure 7D). As shown, the anti-HIS-24 antibody binds with higher affinity to egl-5 and mab-5 genes than the anti-HIL-4 antibody, which is crossreactive to C. elegans linker histone variants [14] ( Figure 7C). Next, to verify the specificity of the HIS-24 binding to Hox genes, we tested the HIS-24 binding to mab-5 gene ectopically expressed in sor-1 background mutation. As previously reported, SOR-1 (together with SOP-2) shares many structural and functional properties with the PRC1 complex, and is involved in the global repression of egl-5 or mab-5 Hox gene expression [25]. As shown, we detected a significantly decreased level of HIS-24 at this region compared to the situation in wild type animals, implicating that HIS-24 enables mab-5 transcriptional repression, thereby influencing its expression ( Figure 7D). Additionally, we observed lower levels of histone H3 occupancy at the mab-5 promoter in sor-1 background mutation than in wild type animals, suggesting that the difference in H3 levels could be due to the nucleosome free region that forms at high levels of expression ( Figure 7D). In addition, mab-5 promoter and intron regions in the his-24 mutant animals showed decreased enrichment of the histone H3 than in wild type animals, suggesting that binding of H3 and HIS-24 can be positively correlated at regulatory regions. In comparison, the H3 changes at 39UTR region of mab-5 in sor-1 and his-24 background mutation were relatively mild than in wild type animals ( Figure 7D).
Unfortunately, we have failed so far to detect HPL-2 at this region using direct ChIP approach.

HIS-24K14me1 and HPL-2 bind H3K27me3 chromatin mark
Recently, we have reported that HIS-24 specifically interacts with H3K27 trimethylated and H3K27 unmodified peptides [18]. While HPL-1 and HPL-2 were able to pull down native HIS-24K14me1, and HPL-2 failed to bind either modified or unmodified HIS-24 peptides in vitro, we asked whether HPL-2 and HIS-24K14me1 repress the transcription of egl-5 and mab-5 genes by binding to H3K27me3 [16,18].
By peptide pull down assay (PD) we observed that HIS-24K14me1 interacts preferentially with H3K27me3 peptide when compared to the unmodified, mono-or di-methylated H3K27 peptides, and conversely, native H3K27me3 binds only the methylated form of HIS-24 peptide ( Figure 9A). Furthermore, we found strong preference of HPL-2 for the trimethylated form of H3K27, as well as for H3K27me2 and H3K9me2/3 as previously reported ( Figure 9A) [16]. No interaction was observed between H3K9me0/1 or H3K27me0/1. We confirmed the results obtained from peptide pull down (PD) by an immunoprecipitation (IP) approach using antibodies raised against different chromatin modification marks and lysates of wild type animals ( Figure 9B). Additionally, we were able to pull down native H3K27me3 using a GFP-antibody directed against GFP-tagged HPL-2 and HIS-24 ( Figure 9C). As a control we used GFP expressed protein under the his-24 promoter to demonstrate the specificity of HPL-2 and HIS-24 binding to H3K27me3 ( Figure 9C). To confirm that HPL-2 and HIS-24 indeed display H3K27me3 binding, we expressed HPL-2 and HIS-24 in E. coli. We did not detect the interaction of HPL-2 with H3K27me3 in contrast to HIS-24, suggesting that additional factors (transcription factors, RNAi machinery, posttranslational modifications of HPL-2) are involved in the mediation of HPL-2 binding to H3K27me3 ( Figure 9D, 9F). In the case of HIS-24 we detected strong preference for H3K27me peptides apart from H3K27me1 ( Figure 9D). The differences in the binding to H3K27me3 between bacterially expressed HIS-24 and native HIS-24 can be explained by the fact that bacterially expressed proteins are not methylated and only the methylated form of HIS-24 binds specifically the H3K27me3. Finally, to exclude that the binding of HPL-2 to H3K27me3 takes place via interaction with the C. elegans HIS-24, we repeated the pull downs using extracts obtained from his-24(ok1024) mutant animals ( Figure 9E). We detected a preference of HPL-2 for H3K27me3 independently of HIS-24 however this binding was reduced compared to binding of HPL-2 to H3K27me3 in the presence of HIS-24 ( Figure 9B, 9E).

HIS-24K14me1 rescues the developmental patterning of the male tail
To assess whether the methylated form of HIS-24 has a causal role in the observed changes of the male tail morphology, we generated his-24::gfp and his-24K14A::gfp transgenic worms in the hpl-2(tm1489); his-24(ok1024) mutant background. We observed that the restoration of HIS-24 levels by expression of HIS-24::GFP rescued the male phenotype and the fused/missing rays were down nearly to zero in the transgenic line ( Figure 10). Importantly, the nonmethylatable HIS-24K14A::GFP mutant failed to rescue the wild type rays development in hpl-2; his-24 animals, suggesting that HIS-24 methylation at lysine 14 is necessary to regulate male tail development ( Figure 10B, 10D, 10E). These results also imply that, at 21uC, hpl-2 and his-24 play a redundant role in the regulation of positional identity in the C. elegans males. Importantly, the analysis of transgene expression at the cellular level by immunostaining and immunoblotting of the rescued hpl-2(tm1489); his-24(ok1024) animals verified that the exogenous HIS-24K14A::GFP mutated form was expressed at a level comparable to that in animals carrying HIS-24::GFP wild type form ( Figure 10C).

Discussion
HP1 and H1 are heterochromatin components that are believed to be associated with global repression of transcriptional activity [4][5]. Surprisingly, our microarray analysis showed that H1 and HP1 play more dynamic and gene-specific roles in the roundworm C. elegans. They grossly affect only a few genes and can have an overlapping function in the same or parallel pathways where they regulate common target genes.
In particular, we found that HIS-24 and HPL-2 can regulate a shared target, the Hox genes. Although, the C. elegans homeobox genes (egl-5, mab-5) are silenced by mechanisms involving H3K27 trimethylation, we showed that the methylated form of HIS-24 and HPL-2 can also serve as essential protein components in establishing and/or maintaining the repressive chromatin state at the selected Hox genes, presumably through their binding to H3K27me3 ( Figure 9F).

Effect of HIS-24 and HPL on gene expression profile and chromatin organization
Our microarray analyses support a role of H1 and HP1 in specific gene regulation, rather than a general repressive function [34][35][36]. Despite global changes in chromatin compaction and synergism of HIS-24 and HPL in aspects of many developmental processes we observed very few and slight changes in gene expression profile of mutants when compared with wild type animals. We detected a set of shared up-and down-regulated genes by HIS-24 and HPL suggesting that redundant roles for HIS-24 and HPL also exist. The relatively small number of regulated genes in observed triple mutant animals may indicate that HPL proteins and HIS-24 serve to fine-tune the regulation of key genes during development or differentiation. This model can be explained by the fact that the sequential arrangement of the linker histone HIS-24 and HPL-2 on the chromatin fibre might influence higher-order chromatin structure and effect nucleosome positioning, and stability [36]. It is possible that the different HPL subtypes and HIS-24 confer subtle differences in the properties of the chromatin fiber which allow for quantitative modulation of gene expression [34,35]. Although the changes in gene transcription are subtle, we think that even 1.5-fold differences in expression can contribute to the marked phenotypic consequences we observed.

Model of transcriptional regulation of egl-5 and mab-5 by HPL-2 and HIS-24K14me1
We demonstrated that HIS-24K14me1, together with HPL-2, has a causal role in transcriptional silencing of egl-5 and mab-5. We propose that HPL-2 and HIS-24K14me1 may serve as essential protein components in establishing and/or maintaining the repressive chromatin state at the selected Hox genes through their interactions with H3K27me3. While we did not observe any phenotypic effects on male tail development either in hpl-2; hpl-1 nor in hpl-1 his-24 background, we speculate that HPL-2 acts redundantly with HIS-24K14me1 to regulate the positional identity in the C. elegans males. Loss of the two heterochromatin components, HIS-24K14me1 and HPL-2, causes significant changes in chromatin structure affecting Hox gene expression in C. elegans. However, since no interaction of HPL-2 and HIS-24K14me1 was observed in immunoprecipitation experiments, it is possible that HPL-2 together with HIS-24K14me1 might be a part of the same protein group involved in the regulation of Hox gene expression. The high degree of redundancy between his-24 and hpl-2 in Hox gene regulation might indicate that these two proteins are the only readers acting in parallel to perform the same role in translating the effects of histone H3K27 trimethylation. However, since we have failed so far to detect HPL-2 at the Hox gene region using direct ChIP approach, it is possible that the mechanisms by which HPL-2 regulates mab-5 and egl-5 might be indirect, involve intermediate factors (RNAi machinery, transcription factors) and depend on an architectural level in the cellular context.

HIS-24K14me1 and HPL-2 as a part of the PcG silencing complex
In mammals, H1 regulates Hox gene activation by promoting DNA demethylation [13]. Although C. elegans does not possess methylated DNA, we speculate that H1 can still influence Hox gene regulation and, together with HPL-2, regulate Hox gene expression as a part of the PcG silencing complex. The interaction of HPL-2 and HIS-24K14me1 with H3K27me3 can regulate the Hox gene in parallel pathway as MES-2 or MES-3, and can be directed to specific parts of the genome. Notably C. elegans HP1/ HPL-2 does not follow the H3K9me2/me3 code [37][38][39][40][41] but it is sufficient to recognize, and to bind H3K27me2/me3. Remarkably, HIS-24 is required for optimal HPL-2 binding to H3K27me3 in vivo.
Interestingly, some PcG proteins containing a chromodomain similar to that found in C. elegans HPL-2 and mammalian HP1s have been shown to bind H3K27me3 [30,42].
Overall, these and our previous results implicate that HPL and HIS-24 share some common functions even though there are Figure 6. mab-5 and egl-5 Hox genes are ectopically expressed in hpl-2; his-24 mutants. (A) A hpl-2; his-24 early L3 mutant male ectopically expresses mab-5::gfp in hypodermal syncytium cell ((hyp7); animal on the left side). Expression of mab-5::gfp in a wild type early L3 male marks very few cells at the posterior (animal on the right side). Scale bar: 25 mm. (B) Quantification of progeny of single and double hpl-2; his-24 mutant hermaphrodites with males carrying mab-5::gfp reporter versus wild type males. (C, D) In a wild type L3 male, expression of egl-5::gfp is limited to the daughters of the ray precursor cells R4, R5 and R6 which give rise to rays 3-6. In hpl-2; his-24 L3 mutant male the reporter is expressed in additional ray sublineages. Scale bar: 25 mm. (E) Quantification of progeny of single and double hpl-2; his-24 mutant hermaphrodites with males carrying egl-5::gfp reporter versus wild type males. (F) Western blot of protein extracts (150 males) from C. elegans wild type, hpl-2; his-24 double mutant and egl-5 transgenic strain probed with antibody raised against EGL-5. Anti-EGL-5 recognized the EGL-5::GFP fusion protein and endogenous EGL-5 in egl-5::gfp transgenic strain. Protein loading was confirmed by probing with an anti-Ce-lamin antibody and western blot stained with Ponceau S. doi:10.1371/journal.pgen.1002940.g006 differences among these proteins [16][17]26]. We conclude that a combination of the H3K27me3 methylation mark, HPL-2 and HIS-24K14me1 could be a major factor in the establishment of stable patterns of selected homeotic gene expression.
The brood size was scored as previously described [14]. All C. elegans strains were maintained at 15uC or at 21uC, unless otherwise specified.
Protein extraction, purification, and identification C. elegans H1 extraction was performed as previously described [16].

Immunofluorescence analysis
Worms from wild type strain and the mutant worms were fixed and stained as previously described [26]. Gonads of worms were stained with fluorescent dye 49,69-diamidino-2-phenylindole (DAPI) diluted 1:1000. The slides were mounted with Vectashield Mounting Medium and analyzed by using Leica DMI 6000 microscope.

Microarray analysis
Microarray analysis from two biological replicates was performed as previously described [16,44]. In brief, 80 to 100 animals in L4 larval stage raised at 21uC were used. The gene expression fold change was calculated from the duplicate microarray data. The fold change cut-off was 1.5 from 2 biological replicates.

Analysis of ray phenotypes
Abnormalities of rays were identified in single, double and triple mutant males in comparison to the wild type worms. Animals were transferred on agar pads (2% agarose) and examined with differential interference contrast (DIC), using Leica DMI 6000 microscope. The number of rays, their position in relation to the anterior-posterior body axis and their shape served as basics of the analysis. Rays which were found outside of their normal formation region were defined as ectopic.

RNA interference (RNAi) experiments
RNAi feeding experiment was performed in 50 mm NGM feeding plates (NGM plates with 100 mg/ml ampicillin, 1 mM IPTG). him-14 (RNAi) (CGC, USA), hpl-1 (RNAi), mes-2 (RNAi) and mes-3 (RNAi) bacterial clones (Sanger Institute, UK) were grown overnight at 37uC in LB medium with 100 mg/ml ampicillin and were spotted onto 50 mm NGM plates. Mixed stage L3 and L4 mutant larval worms were transferred onto feeding plates and incubated at 21uC through several generations. Males were examined on the agar pads using Leica DMI 6000 microscope. Male progeny were scored for the presence of ectopic, under-developed rays and/or ray fusions.
Analysis of EGL-5::GFP and MAB-5::GFP expression in the single, double, and triple mutant strains L3 stage and adult animals from each line were mounted on the agar pads and examined under Leica DMI 6000 microscope. Males were scored for the presence of ectopic EGL-5::GFP or MAB-5::GFP expression.

Peptide pull down analysis
Peptide pull downs were performed as previously described [46]. 10 mg of each biotinylated peptide was coupled to streptavidin-agarose beads (Pierce). For peptide binding experiments following peptides were used: H3 mono-, di-or trimethylated at K9, H3 mono-, di-or trimethylated at K27, H3 unmethylated at K27, HIS-24 monomethylated at K14 and Primer sets for qChIP-PCR are directed to the mab-5 and egl-5 promoters, introns and 39UTR (red bars). (B) qChIP-PCR assay determining HIS-24 occupancy at the mab-5, and egl-5 genes. The results were normalized to binding by anti-HIS-24 antibody in his-24 mutant strains and performed in triplicates. Error bars indicate 6SD (see Table S3). (C) qChIP using anti-HIL-4 antibody and total protein isolated from wild type worms. (D) Decreased level of HIS-24 enrichment at mab-5 using anti-HIS-24 antibody and mab-5::gfp transgenic strain in sor-1 background mutation (KO) as well as wild type (WT) animals. H3 occupancy at mab-5 loci is affected in sor-1 and his-24 background. In sor-1 and his-24 background mab-5 is ectopically expressed in contrast to repressed mab-5 in wild type animals. (C, D) All results were normalized to binding by control IgG antibody and performed in triplicates. Error bars indicate 6SD (see Table S3). doi:10.1371/journal.pgen.1002940.g007 HIS-24 unmethylated at K14. Peptides were generated by Squarix (Germany). Worm extracts were incubated for 2 h at 4uC with the beads (constant rotation). Beads were washed six times with PD 150 buffer (20 mM Hepes pH 7.9, 150 mM KCl, 0.2% Triton-X 100, complete protease inhibitor cocktail, 20% glycerol). Bounded proteins were separated on gradient NuPAGE SDS gel (4-12%).  Western and dot blot C. elegans lysates were prepared and analyzed by western blot as previously described [16,18].

Expression of recombinant HPL-2 protein
The pGEX HPL-2a plasmid (kindly provided by F. Palladino) and HIS-24 pet3a plasmid were expressed in E. coli BL21(DE3) and the recombinant proteins were used for the peptide pull down assay.

Accession numbers
The microarray data can be found in the Gene Expression Omnibus (GEO) of NCBI through accession number GSE33339. Figure S1 The levels of heterochromatin marks are not altered in the hpls, his-24 mutant animals. No changes of the H3K27me3, H3K9me3 and H3 levels were observed in single, double and triple mutant animals. (PDF) Figure S2 Reduced level of HPL-1 after depletion. Reduction of HPL-1 level in hpl-1 depleted his-24; hpl-2 double mutant animals in contrast to his-24; hpl-2 double, where HPL-1 is present. (PDF)