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.     

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

Background  

The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.     

Expression of cis-regulatory mutations of the white locus in metafemales of Drosophila melanogaster.

At the white eye colour locus, there are a number of alleles that have altered expression between males and females. To test these regulatory mutations of the white eye colour locus for their phenotypic expression in metafemales (3X; 2A) compared to diploid females and males, eleven alleles or transduced copies of white were analysed. Two alleles that exhibit dosage compensation between males and females (apricot, blood) also exhibit dosage compensation in metafemales. White-ivory and white-eosin, which fail to dosage compensate in males compared to females, but that are distinct physical lesions, also show a dosage effect in metafemales. Two alleles with greater expression in males than females (spotted, spotted-55) exhibit even lower expression in metafemales. Lastly, five transduced copies of white carrying three different lengths of the white promoter, but that all exhibit higher expression in males, show reduced expression in metafemales, exhibiting an inverse correlation between the level of expression and the dosage of the X chromosome. Because these alleles of white respond to dosage compensation in metafemales as a continuum of the male and female responses, it is concluded that the same basic mechanism of dosage compensation is involved and that the dosage of the X chromosome conditions the sexually dimorphic expression.     

RNA synthesis in Drosophila melanogaster polytene chromosomes indications of simultaneous dosage compensation and dosage effect in X chromosomes

It is shown that the apparent incompleteness of dosage compensation when RNA synthesis is measured autoradiographically is not due to the existence of contiguous dosage compensated and non-dosage compensated genes. Rather this seems to be the result of peculiarities in the coordination of RNA synthesis between the X chromosomes and autosomes. The slope of the line defined by \([\bar X]_i \) and \([\overline {2R} ]_i \) (number of grains over the X and autosomal segments averaged over the different nuclei assayed in each gland) is indistinguishable in males and females (apparent complete dosage compensation). An average of the slopes obtained for different individual glands (from [X] and [2R], the grain counts over each nucleus belonging to a particular gland), on the other hand, has a value in males which is approximately half of the value attained by females (a value of one half, in males, indicates dosage effect since males have one X and females have two).     

X inactivation and disease

X inactivation is the mechanism by which mammals adjust the X-linked gene dosage between the sexes. The dosage difference between XX females and XY males is functionally equalized by silencing one of the two X chromosomes in female cells. This dosage-compensation mechanism is based on the long functional Xist RNA. Here, we review our understanding of dosage compensation and Xist function in the context of disease.     

Possible differences in the two Z chromosomes in male chickens and evolution of MHM sequences in Galliformes

The male hypermethylated (MHM) region of the chicken Z chromosome encodes a non-coding RNA that is expressed only in females. The MHM sequence is found only in galliform birds, and Z genes near this region show an unusual degree of dosage compensation between males and females despite the overall low level of dosage compensation in Z chromosome gene expression in birds. Here we report that the MHM locus shows a dramatic sex difference in the configuration of chromatin, open in females and condensed in males, based on DNA fluorescent in situ hybridization of an MHM probe in interphase nuclei. The demethylating agent 5-aza-cytidine causes an asymmetric effect on the two Z chromosomes of males, altering the chromatin configuration, MHM RNA expression, and H4K16Ac modification, suggesting an inequality in the methylation status and chromatin of the two Z chromosomes. We identified numerous MHM-related genomic and RNA sequences that possess a short conserved sequence common to the majority of clones, suggesting the functional importance of the MHM region. Some of the RNA sequences, which like MHM are expressed in females but not in males, are likely to be polyadenylated and have genomic intron/exon structure. The turkey, another galliform bird, has repetitive sequences in the predicted turkey MHM region, raising the question of regional dosage compensation in the turkey as in the chicken.     

Population Structure of Morphological Traits in Clarkia Dudleyana I. Comparison of F(st) between Allozymes and Morphological Traits

The X-linked white gene when transposed to autosomes retains only partial dosage compensation. One copy of the gene in males expresses more than one copy but less than two copies in females. When inserted in ectopic X chromosome sites, the mini-white gene of the CaspeR vector can be fully dosage compensated and can even achieve hyperdosage compensation, meaning that one copy in males gives more expression than two copies in females. As sequences are removed gradually from the 5' end of the gene, we observe a progressive transition from hyperdosage compensation to full dosage compensation to partial dosage compensation. When the deletion reaches -17, the gene can no longer dosage compensate fully even on the X chromosome. A deletion reaching +173, 4 bp preceeding the AUG initiation codon, further reduces dosage compensation both on the X chromosome and on autosomes. This truncated gene can still partially dosage compensate on autosomes, indicating the presence of dosage compensation determinants in the protein coding region. We conclude that full dosage compensation requires an X chromosome environment and that the white gene contains multiple dosage-compensation determinants, some near the promoter and some in the coding region.     

Modulation of Heterochromatin by Male Specific Lethal Proteins and roX RNA in Drosophila melanogaster Males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila melanogaster males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.     

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.     

All dosage compensation is local: gene-by-gene regulation of sex-biased expression on the chicken Z chromosome

Recent reports have suggested that birds lack a mechanism of wholesale dosage compensation for the Z sex chromosome. This discovery was rather unexpected, as all other animals investigated with chromosomal mechanisms of sex determination have some method to counteract the effects of gene dosage of the dominant sex chromosome in males and females. Despite the lack of a global mechanism of avian dosage compensation, the pattern of gene expression difference between males and females varies a great deal for individual Z-linked genes. This suggests that some genes may be individually dosage compensated, and that some less-than-global pattern of dosage compensation, such as local or temporal, exists on the avian Z chromosome. We used global gene expression profiling in males and females for both somatic and gonadal tissue at several time points in the life cycle of the chicken to assess the pattern of sex-biased gene expression on the Z chromosome. Average fold-change between males and females varied somewhat among tissue time-point combinations, with embryonic brain samples having the smallest gene dosage effects, and adult gonadal tissue having the largest degree of male bias. Overall, there were no neighborhoods of overall dosage compensation along the Z. Taken together, this suggests that dosage compensation is regulated on the Z chromosome entirely on a gene-by-gene level, and can vary during the life cycle and by tissue type. This regulation may be an indication of how critical a given gene's functionality is, as the expression level for essential genes will be tightly regulated in order to avoid perturbing important pathways and networks with differential expression levels in males and females.     

Epigenetic inheritance and genome regulation: is DNA methylation linked to ploidy in haplodiploid insects?

Organisms show great variation in ploidy level. For example, chromosome copy number varies among cells, individuals and species. One particularly widespread example of ploidy variation is found in haplodiploid taxa, wherein males are typically haploid and females are typically diploid. Despite the prevalence of haplodiploidy, the regulatory consequences of having separate haploid and diploid genomes are poorly understood. In particular, it remains unknown whether epigenetic mechanisms contribute to regulatory compensation for genome dosage. To gain greater insights into the importance of epigenetic information to ploidy compensation, we examined DNA methylation differences among diploid queen, diploid worker, haploid male and diploid male Solenopsis invicta fire ants. Surprisingly, we found that morphologically dissimilar diploid males, queens and workers were more similar to one another in terms of DNA methylation than were morphologically similar haploid and diploid males. Moreover, methylation level was positively associated with gene expression for genes that were differentially methylated in haploid and diploid castes. These data demonstrate that intragenic DNA methylation levels differ among individuals of distinct ploidy and are positively associated with levels of gene expression. Thus, these results suggest that epigenetic information may be linked to ploidy compensation in haplodiploid insects. Overall, this study suggests that epigenetic mechanisms may be important to maintaining appropriate patterns of gene regulation in biological systems that differ in genome copy number.     

Dosiskompensation und Dosiseffekt für RNS-Synthese in Chromosomen-Puffs von Drosophila melanogaster

RNA synthesis has been studied autoradiographically in small sections of salivary gland X- and third chromosomes. Grain counts in duplicated and deficient genotypes were compared with those of the same sections in wild types. The main results are: 1. In duplication females the three doses of active loci located in X-chromosome regions 1A-D, 2CD, 2 EF and 3B produce about 50% more RNA than the normal two doses of these regions in wild type females. 2. The double dose of active loci in X-chromosome region 2C-F in duplication males synthesizes approximately 72% more RNA than the normal single dose of this region in wild type males. 3. In duplication females all three homologous loci of regions 1A-D, 2CD and 2EF produce identical amounts of RNA. 4. In females heterozygous for a 3B deficiency the single dose of this region produces nearly as much RNA as the normal two doses in wild type females. 5. Heterozygotes for the deficiency of the active loci 61 E and 61 F produce only half as much RNA in this region as wild types, in both males and females. — The results are discussed with regard to existing hypotheses on dosage compensation.     

Modulation of MSL1 abundance in female Drosophila contributes to the sex specificity of dosage compensation.

Dosage compensation in Drosophila is the mechanism by which X-linked gene expression is made equal in males and females. Proper regulation of this process is critical to the survival of both sexes. Males must turn the male-specific lethal (msl)-mediated pathway of dosage compensation on and females must keep it off. The msl2 gene is the primary target of negative regulation in females. Preventing production of MSL2 protein is sufficient to prevent dosage compensation; however, ectopic expression of MSL2 protein in females is not sufficient to induce an insurmountable level of dosage compensation, suggesting that an additional component is limiting in females. A candidate for this limiting factor is MSL1, because the amount of MSL1 protein in females is reduced compared to males. We have identified two levels of negative regulation of msl1 in females. The predominant regulation is at the level of protein stability, while a second regulatory mechanism functions at the level of protein synthesis. Overcoming these control mechanisms by overexpressing both MSL1 and MSL2 in females results in 100% female-specific lethality.     

Dosage analysis of Z chromosome genes using microarray in silkworm,Bombyx mori

In many organisms, dosage compensation is needed to equalize sex-chromosome gene expression in males and females. Several genes on silkworm Z chromosome were previously detected to show a higher expression level in males and lacked dosage compensation. Whether silkworm lacks global dosage compensation still remains poorly known. Here, we analyzed male:female (M:F) ratios of expression of chromosome-wide Z-linked genes in the silkworm using microarray data. The expression levels of genes on Z chromosome in each tissue were significantly higher in males compared to females, which indicates no global dosage compensation in silkworm. Interestingly, we also found some genes with no bias (M:F ratio: 0.8–1.2) on the Z chromosome. Comparison of male-biased (M:F ratio more than 1.5) and unbiased genes indicated that the two sets of the genes have functional differences. Analysis of gene expression by sex showed that M:F ratios were, to some extent, associated with their expression levels. These results provide useful clues to further understanding roles of dosage of Z chromosome and some Z-linked sexual differences in silkworms.     

The Nature of Quantitative Genetic Variation in Drosophila. III. Mechanism of Dosage Compensation for Sex-Linked Abdominal Bristle Polygenes

Seventeen lines, each homozygous for a different X chromosome but all with a common autosomal genetic background, were constructed and assayed for abdominal bristle number to determine whether dosage compensation operates for sex-linked genes affecting this character.—The regression coefficient of male mean on female mean using a logarithmic scale was 0.90 ± 0.13 and the genetic regression coefficient 0.92, neither differing significantly from unity. The genetic components of variance in males and females were also very similar (0.000234 or 0.000228, respectively). These results indicate that dosage compensation is complete (or nearly so) for sex-linked genes affecting this character. The bristle scores of females did not differ in reciprocal crosses between these lines, thus dosage compensation does not operate by paternal X inactivation.—The question of an adequate scale for abdominal bristle number had to be examined during the study. A logarithmic scale appeared to be adequate for both genotypic and environmental differences.