Regulation of DCC localization by HTZ-1/H2A.Z and DPY-30 does not correlate with H3K4 methylation levels

Dosage compensation is a specialized form of gene regulation that balances sex-chromosome linked gene expression between the sexes. In C. elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC binds along both X chromosomes in hermaphrodites to down-regulate gene expression by half, limiting X-linked gene products to levels produced in XO males. Sequence motifs enriched on the X chromosome play an important role in targeting the DCC to the X. However, these motifs are not strictly X-specific and therefore other factors, such as the chromatin environment of the X chromosome, are likely to aid in DCC targeting. Previously, we found that loss of HTZ-1 results in partial disruption of dosage compensation localization to the X chromosomes. We wanted to know whether other chromatin components coordinated with HTZ-1 to regulate DCC localization. One candidate is DPY-30, a protein known to play a role in DCC localization. DPY-30 homologs in yeast, flies, and mammals are highly conserved members of histone H3 lysine 4 (H3K4) methyltransferase Set1/MLL complexes. Therefore, we investigated the hypothesis that the dosage compensation function of DPY-30 involves H3K4 methylation. We found that in dpy-30 animals the DCC fails to stably bind chromatin. Interestingly, of all the C. elegans homologs of Set1/MLL complex subunits, only DPY-30 is required for stable DCC binding to chromatin. Additionally, loss of H3K4 methylation does not enhance DCC mislocalization in htz-1 animals. We conclude that DPY-30 and HTZ-1 have unique functions in DCC localization, both of which are largely independent of H3K4 methylation.     

Restricting Dosage Compensation Complex Binding to the X Chromosomes by H2A.Z/HTZ-1

Dosage compensation ensures similar levels of X-linked gene products in males (XY or XO) and females (XX), despite their different numbers of X chromosomes. In mammals, flies, and worms, dosage compensation is mediated by a specialized machinery that localizes to one or both of the X chromosomes in one sex resulting in a change in gene expression from the affected X chromosome(s). In mammals and flies, dosage compensation is associated with specific histone posttranslational modifications and replacement with variant histones. Until now, no specific histone modifications or histone variants have been implicated in Caenorhabditis elegans dosage compensation. Taking a candidate approach, we have looked at specific histone modifications and variants on the C. elegans dosage compensated X chromosomes. Using RNAi-based assays, we show that reducing levels of the histone H2A variant, H2A.Z (HTZ-1 in C. elegans), leads to partial disruption of dosage compensation. By immunofluorescence, we have observed that HTZ-1 is under-represented on the dosage compensated X chromosomes, but not on the non-dosage compensated male X chromosome. We find that reduction of HTZ-1 levels by RNA interference (RNAi) and mutation results in only a very modest change in dosage compensation complex protein levels. However, in these animals, the X chromosome–specific localization of the complex is partially disrupted, with some nuclei displaying DCC localization beyond the X chromosome territory. We propose a model in which HTZ-1, directly or indirectly, serves to restrict the dosage compensation complex to the X chromosome by acting as or regulating the activity of an autosomal repellant.     

Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes

Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.     

Identification of X Chromosome Regions in Caenorhabditis Elegans That Contain Sex-Determination Signal Elements

The primary sex-determination signal of Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). This signal coordinately controls both sex determination and X chromosome dosage compensation. To delineate regions of X that contain counted signal elements, we examined the effect on the X/A ratio of changing the dose of specific regions of X, using duplications in XO animals and deficiencies in XX animals. Based on the mutant phenotypes of genes that are controlled by the signal, we expected that increases (in males) or decreases (in hermaphrodites) in the dose of X chromosome elements could cause sex-specific lethality. We isolated duplications and deficiencies of specific X chromosome regions, using strategies that would permit their recovery regardless of whether they affect the signal. We identified a dose-sensitive region at the left end of X that contains X chromosome signal elements. XX hermaphrodites with only one dose of this region have sex determination and dosage compensation defects, and XO males with two doses are more severely affected and die. The hermaphrodite defects are suppressed by a downstream mutation that forces all animals into the XX mode of sex determination and dosage compensation. The male lethality is suppressed by mutations that force all animals into the XO mode of both processes. We were able to subdivide this region into three smaller regions, each of which contains at least one signal element. We propose that the X chromosome component of the sex-determination signal is the dose of a relatively small number of genes.     

Revisiting the X:A signal that specifies Caenorhabditis elegans sexual fate

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.     

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Dosage compensation in Drosophila melanogaster involves the selective targeting of the male X chromosome by the dosage compensation complex (DCC) and the coordinate, ∼2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize ‘chromosomal entry sites’ or ‘high-affinity sites’ may serve such a function. However, to date no DNA binding domain that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2–MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo.     

The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.     

The Dpy-30 Gene Encodes an Essential Component of the Caenorhabditis Elegans Dosage Compensation Machinery

The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X/2A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals.     

Decoding dosage compensation

How the mechanisms of dosage compensation distinguish the sex chromosomes from the autosomes has been something of a mystery. A recent study in Caenorhabditis elegans has identified clusters of two common DNA motifs as a cis-acting code for the recruitment of the DCC, the protein complex that mediates dosage compensation.     

Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.     

Sex Determination in Polyploids of Caenorhabditis Elegans

In Caenorhabditis elegans triploid animals with two X chromosomes (symbolized 3A;2X) are males. However, these triploid males can be feminized by making them mutant for recessive dosage compensation mutations, by adding X chromosome duplications or by microinjecting particular DNA sequences termed feminizing elements. None of these treatments affects diploid males. This study explores several aspects of these treatments in polyploids. The dosage compensation mutants exhibit a strong maternal effect, such that reduction of any of the dosage compensation gene functions in the mother leads to sex reversal of 3A;2X animals. Likewise, all X chromosome duplications tested cause both sex reversal and intersexual development of many 3A;2X animals. Microinjected feminizing element DNA does not cause extensive sex reversal, but does result in intersexual development in 3A;2X animals. Neither X chromosome duplications nor microinjected feminizing elements show the extreme maternal effect of the dosage compensation mutants, although there is indirect evidence for a maternal effect of the feminizing elements. In particular, very little feminizing element DNA needs to be microinjected in order to feminize triploid males, far less than what is needed for stable inheritance, implying that feminizing elements can work within the mother's gonad. However, even very high concentrations of microinjected feminizing elements do not affect sex determination in diploid males, suggesting that they are not part of the numerator of the X/A ratio. In addition, no pair of X chromosome duplications feminizes diploid males, suggesting that none of these duplications contains a numerator of the X/A ratio. Instead, I infer that an X-linked locus, as yet undefined, must be present in two copies for hermaphrodite development to ensue or that the two X chromosomes might interact.