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.     

Dosage compensation in birds

The Z and W sex chromosomes of birds have evolved independently from the mammalian X and Y chromosomes [1]. Unlike mammals, female birds are heterogametic (ZW), while males are homogametic (ZZ). Therefore male birds, like female mammals, carry a double dose of sex-linked genes relative to the other sex. Other animals with nonhomologous sex chromosomes possess "dosage compensation" systems to equalize the expression of sex-linked genes. Dosage compensation occurs in animals as diverse as mammals, insects, and nematodes, although the mechanisms involved differ profoundly [2]. In birds, however, it is widely accepted that dosage compensation does not occur [3-5], and the differential expression of Z-linked genes has been suggested to underlie the avian sex-determination mechanism [6]. Here we show equivalent expression of at least six of nine Z chromosome genes in male and female chick embryos by using real-time quantitative PCR [7]. Only the Z-linked ScII gene, whose ortholog in Caenorhabditis elegans plays a crucial role in dosage compensation [8], escapes compensation by this assay. Our results imply that the majority of Z-linked genes in the chicken are dosage compensated.     

Conservation of Regional Variation in Sex-Specific Sex Chromosome Regulation

Regional variation in sex-specific gene regulation has been observed across sex chromosomes in a range of animals and is often a function of sex chromosome age. The avian Z chromosome exhibits substantial regional variation in sex-specific regulation, where older regions show elevated levels of male-biased expression. Distinct sex-specific regulation also has been observed across the male hypermethylated (MHM) region, which has been suggested to be a region of nascent dosage compensation. Intriguingly, MHM region regulatory features have not been observed in distantly related avian species despite the hypothesis that it is situated within the oldest region of the avian Z chromosome and is therefore orthologous across most birds. This situation contrasts with the conservation of other aspects of regional variation in gene expression observed on the avian sex chromosomes but could be the result of sampling bias. We sampled taxa across the Galloanserae, an avian clade spanning 90 million years, to test whether regional variation in sex-specific gene regulation across the Z chromosome is conserved. We show that the MHM region is conserved across a large portion of the avian phylogeny, together with other sex-specific regulatory features of the avian Z chromosome. Our results from multiple lines of evidence suggest that the sex-specific expression pattern of the MHM region is not consistent with nascent dosage compensation.     

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.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

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.     

Hens,cocks and avian sex determination: A quest for genes on Z or W?

The sex of an individual is generally determined genetically by genes on one of the two sex chromosomes. In mammals, for instance, the presence of the male-specific Y chromosome confers maleness, whereas in Drosophila melanogaster and Caenorhabditis elegans it is the number of X chromosomes that matters. For birds (males ZZ, females ZW), however, the situation remains unclear. The recent discovery that the Z-linked DMRT1 gene, which is conserved across phyla as a gene involved in sexual differentiation, is expressed early in male development suggests that it might be the number of Z chromosomes that regulate sex in birds. On the other hand, the recent identification of the first protein unique to female birds, encoded by the W-linked PKCIW gene, and the observation that it is expressed early in female gonads, suggests that the W chromosome plays a role in avian sexual differentiation. Clearly defining the roles of the DMRT1 and PKC1W genes in gonadal development, and ultimately determining whether avian sex is dependent on Z or W, will require transgenic experiments.     

X染色体的剂量补偿机制

剂量补偿机制(Dosage compensation mechanism)是雌性和雄性X染色体表达平衡的关键,保证两性间由X染色体编码的蛋白质或其他酶类物质在数量上达到平衡。不同生物的剂量补偿机制各不相同,迄今研究表明剂量补偿机制主要有以下3种模式:通过雄性的单个X染色体表达加倍;通过雌性的一条X染色体失活;通过雌性的两个X染色体的表达减半来达到平衡。对剂量补偿的研究有助于揭示X连锁基因的调控机理、性染色体的进化和分化过程,以及解释性染色体畸变的机理,因此,文章将对这种重要的调控机制研究现状及进展进行简要论述。     

SEX-LINKAGE OF SEXUALLY ANTAGONISTIC GENES IS PREDICTED BY FEMALE, BUT NOT MALE, EFFECTS IN BIRDS

Evolutionary theory predicts that sexually antagonistic loci will be preferentially sex-linked, and this association can be empirically testes with data on sex-biased gene expression with the assumption that sex-biased gene expression represents the resolution of past sexual antagonism. However, incomplete dosage compensating mechanisms and meiotic sex chromosome inactivation have hampered efforts to connect expression data to theoretical predictions regarding the genomic distribution of sexually antagonistic loci in a variety of animals. Here we use data on the underlying regulatory mechanism that produce expression sex-bias to test the genomic distribution of sexually antagonistic genes in chicken. Using this approach, which is free from problems associated with the lack of dosage compensation in birds, we show that female-detriment genes are significantly overrepresented on the Z chromosome, and female-benefit genes underrepresented. By contrast, male-effect genes show no over- or underrepresentation on the Z chromosome. These data are consistent with a dominant mode of inheritance for sexually antagonistic genes, in which male-benefit coding mutations are more likely to be fixed on the Z due to stronger male-specific selective pressures. After fixation of male-benefit alleles, regulatory changes in females evolve to minimize antagonism by reducing female expression.     

Triploid plover female provides support for a role of the W chromosome in avian sex determination

Two models, Z Dosage and Dominant W, have been proposed to explain sex determination in birds, in which males are characterized by the presence of two Z chromosomes, and females are hemizygous with a Z and a W chromosome. According to the Z Dosage model, high dosage of a Z-linked gene triggers male development, whereas the Dominant W model postulates that a still unknown W-linked gene triggers female development. Using 33 polymorphic microsatellite markers, we describe a female triploid Kentish plover Charadrius alexandrinus identified by characteristic triallelic genotypes at 14 autosomal markers that produced viable diploid offspring. Chromatogram analysis showed that the sex chromosome composition of this female was ZZW. Together with two previously described ZZW female birds, our results suggest a prominent role for a female determining gene on the W chromosome. These results imply that avian sex determination is more dynamic and complex than currently envisioned.     

棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析

【目的】棉铃虫Helicoverpa armigera的剂量补偿(dosage compensation, DC)分子机制尚不清楚。本研究旨在通过克隆棉铃虫雄性特异性致死(male specific lethal, msl) 基因Hamsl1,利用RNA干扰技术明确其是否参与调控棉铃虫剂量补偿。【方法】利用RT-PCR同源克隆棉铃虫Hamsl1基因全长cDNA; 利用qPCR技术研究Hamsl1基因在棉铃虫不同发育时期的表达谱;通过显微注射Hamsl1 siRNA到棉铃虫3龄幼虫中对Hamsl1基因进行RNA干扰后,利用qPCR技术检测15个Z染色体基因的表达情况,分析Hamsl1是否调控Z染色体基因剂量。【结果】成功克隆了棉铃虫Hamsl1基因的cDNA序列,鉴定出Hamsl1基因mRNA存在2种剪接体,分别命名为Hamsl1a(GenBank登录号: MK564008)和Hamsl1b(GenBank登录号: MK564009)。功能域分析发现HaMSL1含有典型的PEHE和coiled-coil功能域,具有MSL1蛋白的特征。qPCR分析表明,Hamsl1基因位于棉铃虫Z染色体上;棉铃虫Hamsl1a与Hamsl1b基因表达均具有发育时期特异性,在成虫期表达量最高,且雌雄化蛹后基因表达量差异显著,具有性别特异性。通过同源比对和qPCR分析,在DNA水平鉴定了15个Z染色体候选基因。显微注射Hamsl1 siRNA于3龄幼虫体内72 h,干扰效率为36.01%~64.27%,并未发生雄性致死现象;与对照组相比,Hamsl1 RNAi处理组中棉铃虫15个Z染色体基因在雄性个体中整体呈现表达量上调趋势,而在雌性个体中平均表达水平差异不显著。【结论】本研究初步探明Hamsl1基因位于棉铃虫Z染色体上,且该基因可能通过抑制雄性棉铃虫Z染色体基因表达,调控棉铃虫Z染色体剂量补偿。本研究为深入研究棉铃虫剂量补偿分子机制和绿色防控棉铃虫提供了理论基础。