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Showing papers on "Dosage compensation published in 2003"


Journal ArticleDOI
TL;DR: Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysines 27 on Xi and that this, in turn, is needed to stabilize the Xi chromatin structure.

678 citations


Journal ArticleDOI
31 Jan 2003-Science
TL;DR: Using comparative genomics, it is found that the X chromosome is a disfavored location for genes selectively expressed in males in Drosophila melanogaster and these same X-chromosome genes are exceptionally poorly conserved in the mosquito Anopheles gambiae.
Abstract: Sex chromosomes are primary determinants of sexual dimorphism in many organisms. These chromosomes are thought to arise via the divergence of an ancestral autosome pair and are almost certainly influenced by differing selection in males and females. Exploring how sex chromosomes differ from autosomes is highly amenable to genomic analysis. We examined global gene expression in Drosophila melanogaster and report a dramatic underrepresentation of X-chromosome genes showing high relative expression in males. Using comparative genomics, we find that these same X-chromosome genes are exceptionally poorly conserved in the mosquito Anopheles gambiae. These data indicate that the X chromosome is a disfavored location for genes selectively expressed in males.

519 citations


Journal ArticleDOI
18 Dec 2003-Nature
TL;DR: It is argued that the XX embryo is in fact dosage compensated at conception along much of the X chromosome, and proposed that imprinted X inactivation results from inheritance of a pre-inactivated X chromosome from the paternal germ line.
Abstract: In mammals, dosage compensation ensures equal X-chromosome expression between males (XY) and females (XX) by transcriptionally silencing one X chromosome in XX embryos In the prevailing view, the XX zygote inherits two active X chromosomes, one each from the mother and father, and X inactivation does not occur until after implantation Here, we report evidence to the contrary in mice We find that one X chromosome is already silent at zygotic gene activation (2-cell stage) This X chromosome is paternal in origin and exhibits a gradient of silencing Genes close to the X-inactivation centre show the greatest degree of inactivation, whereas more distal genes show variable inactivation and can partially escape silencing After implantation, imprinted silencing in extraembryonic tissues becomes globalized and more complete on a gene-by-gene basis These results argue that the XX embryo is in fact dosage compensated at conception along much of the X chromosome We propose that imprinted X inactivation results from inheritance of a pre-inactivated X chromosome from the paternal germ line

366 citations


Journal ArticleDOI
TL;DR: Common silencing mechanisms are employed by the various imprinting domains, including silencer elements that nucleate and propagate a silent chromatin state, insulator elements that prevent promoter-enhancer interactions when hypomethylated on one parental allele, and antisense RNAs that function in silencing the overlapping sense gene and more distantly located genes.
Abstract: An intriguing characteristic of imprinted genes is that they often cluster in large chromosomal domains, raising the possibility that gene-specific and domain-specific mechanisms regulate imprinting. Several common features emerged from comparative analysis of four imprinted domains in mice and humans: (a) Certain genes appear to be imprinted by secondary events, possibly indicating a lack of gene-specific imprinting marks; (b) some genes appear to resist silencing, predicting the presence of cis-elements that oppose domain-specific imprinting control; (c) the nature of the imprinting mark remains incompletely understood. In addition, common silencing mechanisms are employed by the various imprinting domains, including silencer elements that nucleate and propagate a silent chromatin state, insulator elements that prevent promoter-enhancer interactions when hypomethylated on one parental allele, and antisense RNAs that function in silencing the overlapping sense gene and more distantly located genes. These commonalities are reminiscent of the behavior of genes subjected to, and the mechanisms employed in, dosage compensation.

266 citations


Journal ArticleDOI
01 Dec 2003-Genetics
TL;DR: The hypothesis that epigenetic loss of gene repression occurs in normal tissues and may be a contributing factor in progressive physiological dysfunction seen during mammalian aging is supported.
Abstract: Epigenetic control of gene expression is a consistent feature of differentiated mammalian cell types. Epigenetic expression patterns are mitotically heritable and are stably maintained in adult cells. However, unlike somatic DNA mutation, little is known about the occurrence of epigenetic change, or epimutation, during normal adult life. We have monitored the age-associated maintenance of two epigenetic systems--X inactivation and genomic imprinting--using the genes Atp7a and Igf2, respectively. Quantitative measurements of RNA transcripts from the inactive and active alleles were performed in mice from 2 to 24 months of age. For both genes, older animal cohorts showed reproducible increases in transcripts expressed from the silenced alleles. Loss of X chromosome silencing showed cohort mean increases of up to 2.2%, while imprinted-gene activation increased up to 6.7%. The results support the hypothesis that epigenetic loss of gene repression occurs in normal tissues and may be a contributing factor in progressive physiological dysfunction seen during mammalian aging. Quantitatively, the loss of epigenetic control may be one to two orders of magnitude greater than previously determined somatic DNA mutation.

186 citations


Book ChapterDOI
TL;DR: The initial step in mammalian sexual differentiation is based on the XX: XY chromosomal system, and a single X chromosome is active in the female soma so as to eliminate gross aneuploidy effects between males and females; this is the broad outline of mammalian X-chromosome regulation.
Abstract: The initial step in mammalian sexual differentiation is based on the XX: XY chromosomal system. In order to function properly, this chromosomal mechanism must be regulated to eliminate the aneuploidy effects in somatic tissues and still insure normal sexual differentiation and development. In mammalian forms, an X-chromosome regulatory mechanism has evolved to carry out these developmental functions. The two X chromosomes in the female germ line remain active through most of their ontogeny to bring about normal ovarian function; a single X chromosome is active in the female soma so as to eliminate gross aneuploidy effects between males and females; and in the male germ line the single X chromosome is inactivated or eliminated at an apparently critical stage in spermiogenesis. This is the broad outline of mammalian X-chromosome regulation. The specifics vary in different forms: random X-chromosome inactivation in most eutherian mammals, a possible nonrandom mechanism in marsupials, and a chromosomal elimination system in the creeping vole, Micron’s oregoni.

184 citations


Journal ArticleDOI
TL;DR: The existing data concerning Sxl protein, its biological functions, and the regulation of its target genes are reviewed.
Abstract: In the past two decades, scientists have elucidated the molecular mechanisms behind Drosophila sex determination and dosage compensation. These two processes are controlled essentially by two different sets of genes, which have in common a master regulatory gene, Sex-lethal (Sxl). Sxl encodes one of the best-characterized members of the family of RNA binding proteins. The analysis of different mechanisms involved in the regulation of the three identified Sxl target genes (Sex-lethal itself, transformer, and male specific lethal-2) has contributed to a better understanding of translation repression, as well as constitutive and alternative splicing. Studies using the Drosophila system have identified the features of the protein that contribute to its target specificity and regulatory functions. In this article, we review the existing data concerning Sxl protein, its biological functions, and the regulation of its target genes.

170 citations


Journal ArticleDOI
TL;DR: The need for X-linked dosage compensation was a major driving force in the evolution of genomic imprinting in mammals and it is proposed that imprinting was first fixed on the X chromosome for XCI and subsequently acquired by autosomes.

117 citations


Journal ArticleDOI
TL;DR: It is shown that tissue-specific genes tend to be more abundant on the human X chromosome, and that, controlling for this effect, genes expressed exclusively in prostate are enriched on thehuman X chromosome; this is consistent with Rice's model of the evolution of sexually antagonistic alleles.
Abstract: There is increasing evidence that X chromosomes have an unusual complement of genes, especially genes that have sex-specific expression. However, whereas in worm and fly the X chromosome has a dearth of male-specific genes, in mice genes that are uniquely expressed in spermatogonia are especially abundant on the X chromosome. Is this latter enrichment true for nongermline, male-specific genes in mammals, and is it found also for female-specific genes? Here, using SAGE data, we show (1) that tissue-specific genes tend to be more abundant on the human X chromosome, (2) that, controlling for this effect, genes expressed exclusively in prostate are enriched on the human X chromosome, and (3) that genes expressed exclusively in mammary gland and ovary are not so enriched. This we propose is consistent with Rice's model of the evolution of sexually antagonistic alleles.

112 citations


Journal ArticleDOI
TL;DR: These results support a model for distribution of MSL complexes, in which local spreading in cis from roX genes is balanced with diffusion of soluble complexes in trans, by nucleation of spreading from their sites of synthesis.
Abstract: MSL proteins and noncoding roX RNAs form complexes to up-regulate hundreds of genes on the Drosophila male X chromosome, and make X-linked gene expression equal in males and females. Altering the ratio of MSL proteins to roX RNA dramatically changes X-chromosome morphology. In protein excess, the MSL complex concentrates near sites of roX transcription and is depleted elsewhere. These results support a model for distribution of MSL complexes, in which local spreading in cis from roX genes is balanced with diffusion of soluble complexes in trans. When overexpressed, MSL proteins can recognize the X chromosome, modify histones, and partially restore male viability even in the absence of roX RNAs. Thus, the protein components can carry out all essential functions of dosage compensation, but roX RNAs facilitate the correct targeting of MSL complexes, in part by nucleation of spreading from their sites of synthesis.

109 citations


Journal ArticleDOI
TL;DR: A digenic model in which the presence of a "mutated" allele in a second gene, leading to a less functional protein, determines the clinical severity of the MECP2 mutation is proposed, supported by the identification of the same mutation in XLMR and RTT cases.
Abstract: Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively girls. It is currently considered a monogenic X-linked dominant disorder due to mutations in MECP2 gene, encoding the methyl-CpG binding protein 2. A few RTT male cases, resulting from mosaicism for MECP2 mutations, have been reported. Male germline MECP2 mutations cause either severe encephalopathy with death at birth (usually in brothers of classical RTT females) or X-linked recessive mental retardation (XLMR). To date the wide phenotypic heterogeneity associated with MECP2 mutations in females (from classical RTT to healthy carriers) has been explained by differences in X chromosome inactivation. However, conflicting results have been obtained in different studies, with both random and highly skewed X-inactivation reported in healthy carrier females. Consequently it is possible that mechanisms other than X-inactivation play a role in the expressivity of MECP2 mutations. To explain the phenotypic heterogeneity associated with MECP2 mutations we propose a digenic model in which the presence of a "mutated" allele in a second gene, leading to a less functional protein, determines the clinical severity of the MECP2 mutation. The model is supported by the identification of the same mutation in XLMR and RTT cases. The carrier mothers of XLMR families are clinically asymptomatic and present balanced X chromosome inactivation. Therefore the same mutation arising in different genetic backgrounds can cause XLMR in males, remain silent in the carrier females and cause classic RTT in females. MECP2 mutations account for approximately 70–80% of classic RTT cases. MECP2 negative cases might result from mutations in noncoding regions of MECP2 gene. Alternatively, these cases might be due to mutations in other genes (locus heterogeneity). This hypothesis is supported by the identification of several chromosomal rearrangements in MECP2 negative patients with RTT and RTT-like phenotypes. MeCP2 is considered a general transcriptional repressor. However, conditional mouse mutants with selective loss of Mecp2 in the brain develop clinical manifestations similar to RTT, indicating that MECP2 is exclusively required for central nervous system function. The involvement of MeCP2 in methylation-specific transcriptional repression suggests that MECP2 related disorders result from dysregulated gene expression. Studies on gene expression have been performed in mouse and human brains. A relatively small number of gene expression changes were identified. It is possible that MeCP2 causes dysregulation of a very small subset of genes that are not detected with this method of analysis, or that very subtle changes in many genes cause the neuronal phenotype.

Journal ArticleDOI
TL;DR: Cells from ICF patients who are deficient in one of the DNA methyltransferases, DNMT3B, provide an opportunity to explore and refine the proposed hypothesis that long interspersed nuclear element 1 (LINE-1 or L1) repeats may be mediators for the spread of X chromosome inactivation.
Abstract: Lyon has proposed that long interspersed nuclear element 1 (LINE-1 or L1) repeats may be mediators for the spread of X chromosome inactivation. Cells from ICF patients who are deficient in one of the DNA methyltransferases, DNMT3B, provide an opportunity to explore and refine this hypothesis. Southern blot and bisulfite methylation analyses indicate that, in normal somatic cells, X-linked L1s are hypermethylated on both the active and inactive X chromosomes. In contrast, ICF syndrome cells with DNMT3B mutations have L1s that are hypomethylated on the inactive X, but not on the active X or autosomes. The DNMT3B methyltransferase, therefore, is required for methylation of L1 CpG islands on the inactive X, whereas methylation of the corresponding L1 loci on the active X, as well as most autosomal L1s, is accomplished by another DNA methyltransferase. This unique phenomenon of identical allelic modifications by different enzymes has not been previously observed. Apart from CpG island methylation, the ICF inactive X is basically normal in that it forms a Barr body, is associated with XIST RNA, mostly replicates late, and its X-inactivated genes are mostly silent. Because the unmethylated state of the ICF inactive X L1s probably reflects their methylation status at the time of X inactivation, these data suggest that unmethylated L1 elements, but not methylated L1s, may have a role in the spreading of X chromosome inactivation.

Journal ArticleDOI
TL;DR: Wild-type MSL complex titers are critical for correct targeting to the X chromosome in Drosophila, and support a model in whichMSL complex binding to theX is directed by a hierarchy of target sites that display different affinities for the MSL proteins.
Abstract: In Drosophila, dosage compensation requires assembly of the Male Specific Lethal (MSL) protein complex for doubling transcription of most X-linked genes in males. The recognition of the X chromosome by the MSL complex has been suggested to include initial assembly at ~35 chromatin entry sites and subsequent spreading of mature complexes in cis to numerous additional sites along the chromosome. To understand this process further we examined MSL patterns in a range of wild-type and mutant backgrounds producing different amounts of MSL components. Our data support a model in which MSL complex binding to the X is directed by a hierarchy of target sites that display different affinities for the MSL proteins. Chromatin entry sites differ in their ability to provide local intensive binding of complexes to adjacent regions, and need high MSL complex titers to achieve this. We also mapped a set of definite autosomal regions (~70) competent to associate with the functional MSL complex in wild-type males. Overexpression of both MSL1 and MSL2 stabilizes this binding and results in inappropriate MSL binding to the chromocenter and the 4th chromosome. Thus, wild-type MSL complex titers are critical for correct targeting to the X chromosome.

Journal ArticleDOI
TL;DR: It is proposed that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome.

Journal ArticleDOI
TL;DR: A 110 bp sequence in roX2 is identified characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species.

Journal ArticleDOI
TL;DR: It is demonstrated that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA.

Journal ArticleDOI
TL;DR: Analysis of a series of targeted mutations at the 5' end of the Xist locus indicates that X chromosome choice is determined by the balance of Xist sense and antisense transcription prior to the onset of random X inactivation.
Abstract: The X-inactive-specific transcript (Xist) locus is a cis-acting switch that regulates X chromosome inactivation in mammals. Over recent years an important goal has been to understand how Xist is regulated at the initiation of X inactivation. Here we report the analysis of a series of targeted mutations at the 5' end of the Xist locus. A number of these mutations were found to cause preferential inactivation, to varying degrees, of the X chromosome bearing the targeted allele in XX heterozygotes. This phenotype is similar to that seen with mutations that ablate Tsix, an antisense RNA initiated 3' of Xist. Interestingly, each of the 5' mutations causing nonrandom X inactivation was found to exhibit ectopic sense transcription in embryonic stem (ES) cells. The level of ectopic transcription was seen to correlate with the degree of X inactivation skewing. Conversely, targeted mutations which did not affect randomness of X inactivation also did not exhibit ectopic sense transcription. These results indicate that X chromosome choice is determined by the balance of Xist sense and antisense transcription prior to the onset of random X inactivation.

Journal ArticleDOI
TL;DR: The results indicate that, in contrast to other chromatin-remodeling complexes that enhance transcription, the MSL complex targets active chromatin.
Abstract: The male-specific lethal (MSL) complex of Drosophila is responsible for the presence of a monoacetylated isoform of histone H4 (H4Ac16), found exclusively on the X chromosome of males. This particular covalent modification of histone H4 is correlated with a 2-fold enhancement of the transcription of most X-linked genes in Drosophila males, which is the basis of dosage compensation in this organism. Although widespread along the X chromosome, the MSL complex is not distributed uniformly, as can be seen by the indirect cytoimmunofluorescence staining of larval salivary-gland polytene chromosomes. This distribution pattern has been interpreted as a reflection of the tissue-specific transcriptional activity of the larval salivary gland and as an indication that the MSL complex associates with active chromatin. We have tested this hypothesis by comparing the chromosomal distribution of the complex in two different tissues. We performed this comparison by following the pattern of association of the complex at a specific site on salivary-gland chromosomes during larval development and determining whether an ectopic promoter located in a complex-devoid region of the X chromosome is able to attract the complex upon activation. Our results indicate that, in contrast to other chromatin-remodeling complexes that enhance transcription, the MSL complex targets active chromatin.

Journal ArticleDOI
TL;DR: It is likely that there is a much more complex interplay between the different features which leads to the extremely stable silencing observed in female somatic cells.
Abstract: Compensating for the dosage difference in X-linked genes between male and female mammals involves the formation of an extremely stable heterochromatin structure on one of the two X chromosomes in females. The inactive X acquires numerous features of silent chromatin, including the expression of a noncoding RNA, a switch to late replication, histone modifications, recruitment of the histone variant macroH2A and DNA hypermethylation. Although the induction of inactivation in differentiating mouse embryonic stem cells suggests that the onset of each of these features appears to occur in a sequential manner, it is likely that there is a much more complex interplay between the different features which leads to the extremely stable silencing observed in female somatic cells. Expression of the untranslated RNA, XIST, is required in cis for the establishment of the heterochromatic state. Recent results have started to elucidate how expression of Xist is controlled, including the role of the antisense transcript Tsix.

Journal ArticleDOI
01 Jul 2003-Genetics
TL;DR: It was found that deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy in Drosophila melanogaster males, and disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed.
Abstract: Drosophila melanogaster males dosage compensate by twofold upregulation of the expression of genes on their single X chromosome. This process requires at least five proteins and two noncoding RNAs, roX1 and roX2, which paint the male X chromosome. We used a deletion analysis to search for functional RNA domains within roX1, assaying RNA stability, targeting of the MSL proteins to the X, and rescue of male viability in a roX1(-) roX2(-) mutant background. We found that deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy. The 3' 600 nt of roX1 were most sensitive to mutations, affecting proper localization and 3' processing of the RNA. Disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed.

Journal ArticleDOI
TL;DR: The roX1 and roX2 genes of Drosophila produce non-coding transcripts that localize to the X-chromosome, which support a model for the ordered assembly of the complex in embryos.

Journal ArticleDOI
TL;DR: Dpy-21 mutations, shown here to be null, cause elevated X-linked gene expression in XX animals, but unlike mutations in other dosage compensation genes, they do not cause extensive XX-specific lethality or disrupt the stability or targeting of the dosage compensation complex to X.
Abstract: In C. elegans, an X-chromosome-wide regulatory process compensates for the difference in X-linked gene dose between males (XO) and hermaphrodites (XX) by equalizing levels of X-chromosome transcripts between the sexes. To achieve dosage compensation, a large protein complex is targeted to the X chromosomes of hermaphrodites to reduce their expression by half. This repression complex is also targeted to a single autosomal gene, her-1. By silencing this male-specific gene, the complex induces hermaphrodite sexual development. Our analysis of the atypical dosage compensation gene dpy-21 revealed the first molecular differences in the complex that achieves gene-specific versus chromosome-wide repression. dpy-21 mutations, shown here to be null, cause elevated X-linked gene expression in XX animals, but unlike mutations in other dosage compensation genes, they do not cause extensive XX-specific lethality or disrupt the stability or targeting of the dosage compensation complex to X. Nonetheless, DPY-21 is a member of the dosage compensation complex and localizes to X chromosomes in a hermaphrodite-specific manner. However, DPY-21 is the first member of the dosage compensation complex that does not also associate with her-1. In addition to a difference in the composition of the complex at her-1 versus X, we also found differences in the targeting of the complex to these sites. Within the complex, SDC-2 plays the lead role in recognizing X-chromosome targets, while SDC-3 plays the lead in recognizing her-1 targets.

Journal ArticleDOI
TL;DR: It is proposed that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females.
Abstract: Birds show female heterogamety, with ZZ males and ZW females. It is still not clear whether the W is female-determining, or whether two doses of the Z chromosomes are male-determining, or both. This question could easily be settled by the sexual phenotypes of ZZW and ZO birds, in the same way that the sexual phenotypes of XXY and XO showed that the Y is male determining in humans, but that the dosage of an X-borne gene determines sex in Drosophila. However, despite extensive searches, no ZZW or ZO diploid birds have been satisfactorily documented, so we must assume that these genotypes are embryonic lethals. Given that ZW and ZZ are viable and the W contains few genes it is not clear why this should be so. Here I propose that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females. ZO birds would therefore have only half the normal dose of Z-linked gene product and ZZW would have twice the amount, both of which would undoubtedly be incompatible with life. Reports of other aneuploids and triploids are also consistent with this hypothesis.

Journal ArticleDOI
TL;DR: Using the completed genomic sequences of mouse and human, comparative analyses of imprinted genes and gene clusters indicate that imprinting clusters may have been linked together on one (or a few) ancestral pre-imprinted chromosome(s), arguing for a common mechanistic origin of imprinting control.
Abstract: Using the completed genomic sequences of mouse and human we performed a comparative analyses of imprinted genes and gene clusters. For many imprinted genes we could detect imprinted as well as non-imprinted paralogues. The inter- and intrachromosomal similarities between paralogues and their linkage to imprinting clusters suggests that imprinted genes were dispersed throughout the genome by gene duplications as well as translocation and transposition events. Our findings indicate that imprinting clusters may have been linked together on one (or a few) ancestral pre-imprinted chromosome(s), arguing for a common mechanistic origin of imprinting control. Imprinting may originally have evolved on a simple basis of dosage compensation required for some duplicated genes (chromosomes) followed by selection of sex-biased expression control.

Book ChapterDOI
TL;DR: The protein Sex-lethal acts as a master regulatory switch, being expressed exclusively in female flies and inducing female-specific patterns of alternative splicing on target genes, which control somatic and germline sexual differentiation, sexual behavior and X chromosome dosage compensation.
Abstract: Posttranscriptional regulation is of fundamental importance for establishing the gene expression programs that determine sexual identity in the fruitfly Drosophila melanogaster. The protein Sex-lethal acts as a master regulatory switch, being expressed exclusively in female flies and inducing female-specific patterns of alternative splicing on target genes. As a consequence, other regulatory factors are expressed in a sex-specific manner, and these factors control somatic and germline sexual differentiation, sexual behavior and X chromosome dosage compensation. Here, we review the molecular mechanisms responsible for splicing regulation in Drosophila sexual determination.

Book ChapterDOI
TL;DR: This chapter outlines various techniques for analyzing X-inactivation kinetics in differentiating Embryonic stem (ES) cells, particularly on changing patterns of histone modifications during X inactivation, using immunuofluorescence combined with RNA fluorescence in situ hybridization (FISH) on interphase nuclei, as well as metaphase chromosome staining combined with DNA FISH.
Abstract: Publisher Summary This chapter outlines various techniques for analyzing X-inactivation kinetics in differentiating Embryonic stem (ES) cells. The chapter focuses particularly on changing patterns of histone modifications during X inactivation, using immunuofluorescence combined with RNA fluorescence in situ hybridization (FISH) on interphase nuclei, as well as metaphase chromosome staining combined with DNA FISH. X-chromosome inactivation provides a powerful model system to investigate the different steps in facultative heterochromatin formation. During early development, one of the two X chromosomes is transcriptionally silenced in every cell of a female embryo, thereby achieving dosage compensation between males and females for X-linked gene products. The X inactivation process is dependent on the action of a unique RNA, Xist, which coats the X chromosome in cis and induces its inactivation. Once established, the inactive state of the X chromosome is highly stable in somatic cells and is normally only reversed in the female germ line. In addition, the chapter also describes a protocol for assaying late replication timing of the inactive X chromosome. Using these techniques it is possible to determine the relative order of the following events: Xist RNA coating occurs within the first 24–48 hours of differentiation; histone H3 modifications, such as hypomethylation of Lys-4, hypoacetylation of Lys- 9, and hypermethylation of Lys-9 and Lys-27 are detectable on the X chromosome in a proportion of interphase cells as soon as Xist RNA accumulates.

Journal ArticleDOI
TL;DR: An unanticipated role for GHMP kinase family members as mediators of sexual differentiation and dosage compensation and, possibly, other aspects of differentiation and development is demonstrated.
Abstract: Sex determination is the critical and universal developmental pathway underlying sexual reproduction. Its manifestations are pervasive and often conspicuous. Whereas the presence or absence of the Y chromosome dictates male or female development in mammals, sexual fate in the fruit fly Drosophila melanogaster and the free-living nematode Caenorhabditis elegans is determined genetically by the number of X chromosomes relative to the number of sets of autosomes. In mammals, the primary sex determining gene is SRY, which is present only on the Y chromosome and encodes an HMG domain-containing transcription factor. In the fruit fly, the primary sex determination gene Sex-lethal (Sxl; Maine et al. 1985) is a female-specific trans-acting gene regulator that binds tra transcripts and directs alternative splicing (Inoue et al. 1990). The SRY (Werner et al. 1995) and SXL (Handa et al. 1999) interactions with polynucleotides have been characterized structurally. In C. elegans, sexual differentiation is regulated by the expression levels of the developmental switch gene xol-1. High and low levels of xol-1 result in male (XO) and hermaphrodite (XX) development (Fig. ​(Fig.1),1), respectively. XOL-1 activity is absolutely required for proper sexual differentiation and male viability (Rhind et al. 1995), but its mechanism of action is unknown. Figure 1 Genetic control of sex determination and dosage compensation in C. elegans. xol-1 is the primary sex-determination switch gene and the direct molecular target of the X-chromosome counting mechanism. (Top) Male (XO). High xol-1 activity specifies male ... The cooperative activity of at least four X-linked genes, termed X-signal elements, represses expression of xol-1 (for review, see Meyer 2000a). By doubling the number of X-signal elements, an XX embryo reduces xol-1 expression by ∼10-fold (Rhind et al. 1995), facilitating hermaphrodite development. Two C. elegans X-signal elements have been characterized molecularly as follows: FOX-1 (Hodgkin et al. 1994; Nicoll et al. 1997; Skipper et al. 1999), an RNA-binding protein that may regulate alternate splicing of xol-1 RNA, and SEX-1 (Carmi et al. 1998), a nuclear receptor and likely a transcription factor. Although hermaphrodites and males differ in X chromosome number, the expression of most X-linked genes must be equal to ensure viability. This is accomplished through dosage compensation, which reduces expression of X-linked genes in hermaphrodites to male levels (for reviews, see Wood et al. 1997; Hansen and Pilgrim 1999; Meyer 2000b; Boag et al. 2001). High levels of XOL-1 in males correlate with low SDC-2 expression, preventing dosage compensation (Miller et al. 1988; Rhind et al. 1995). Conversely, low levels of XOL-1 in hermaphrodites correlate with high SDC-2 expression and the assembly on the X chromosome of the dosage compensation complex, which is composed of sdc, dpy, and mix-1 gene products (Nonet and Meyer 1991; Chuang et al. 1996; Lieb et al. 1996, 1998; Davis and Meyer 1997; Dawes et al. 1999; Chu et al. 2002). Other genes downstream of xol-1, such as her, tra, and fem, whose activities are inversely related in hermaphrodites and males, coordinate sexual differentiation (Goodwin and Ellis 2002). Null mutants of xol-1 are XO-lethal, inappropriately activating dosage compensation where only one X chromosome is present, whereas XOL-1 overexpression is XX-lethal, deactivating the dosage compensation pathway and elevating the expression of X chromosome genes to lethal levels in hermaphrodites (Rhind et al. 1995). XOL-1 is an acidic 51-kD nuclear protein (pKa 4.6), whose transcript is expressed at high levels only in pre-comma stage XO embryos (Rhind et al. 1995). XOL-1 transcripts are present in low levels throughout other larval stages in XO animals, but are nearly undetectable in XX larvae and adults of both sexes (Rhind et al. 1995). Currently, XOL-1 is annotated as a subtilisin-like protease on the basis of primary sequence (http://www.wormbase.org). BLAST searches of Genbank failed to identify any homologs that may have provided additional clues as to the function of XOL-1. Thus, we used x-ray crystallography to investigate the function of XOL-1, hypothesizing that the three-dimensional structure of the protein would yield insights into its nature, largely uncharacterized biochemically. The resulting crystal structure of XOL-1 (Fig. ​(Fig.2A)2A) unambiguously and unexpectedly defines XOL-1 as a member of the GHMP kinase family, a family of proteins known to be involved in small molecule metabolism, but not known to participate directly in sexual differentiation or dosage compensation. Figure 2 Comparison of XOL-1 and GHMP kinase structures. (A) The structure of XOL-1. Ribbon diagram of XOL-1 (PDB ID: 1MG7). Domain 1 consists of β-strands 2–7 and 12 (cyan) and α-helices 1–5 (yellow). Domain 2 consists of β-strands ... Galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase were originally identified as prototypic members of the GHMP kinase family and observed to contain a conserved Pro–Xaa3–Gly–Leu–Gly–Ser–Ser–Ala–Ala motif (Fig, 3A) that was hypothesized (Tsay and Robinson 1991; Bork et al. 1993), and later proved (Zhou et al. 2000; Krishna et al. 2001; Fu et al. 2002) to be involved in ATP binding. The nucleotide fold of GHMP kinases is distinct from those of other kinases, that is, P-loop, protein, and Hsp70-like kinases (Zhou et al. 2000; Bonanno et al. 2001; Fu et al. 2002; Romanowski et al. 2002; Yang et al. 2002), and binds ATP in both syn and anti-conformations (Zhou et al. 2000; Fu et al. 2002). GHMP kinases are found in bacteria, archaea, and eukaryotes and contain an unusual left-handed β–α–β fold similar to that observed in domain IV of elongation factor G (Zhou et al. 2000). In humans, deficiency of galactokinase, which participates in the conversion of galactose to glucose, contributes to cataract formation (Monteleone et al. 1971; Beutler 1972; Harley et al. 1972; Levy et al. 1972). Mutations in mevalonate kinase, an enzyme involved in the synthesis of sterols from acetate, are associated with mevalonic aciduria (Hoffmann et al. 1986; Schafer et al. 1992; Houten et al. 1999b) and hyperimmunoglobulinemia D/periodic fever syndrome (Drenth et al. 1999; Houten et al. 1999a, 2001; Cuisset et al. 2001; Rios et al. 2001; Simon et al. 2001). To date, no GHMP kinases have been shown to function in developmental pathways unrelated to metabolism.

Journal ArticleDOI
TL;DR: The focus of this review is on large RNAs that act in the dosage-compensation pathways of flies and mammals that do not appear to act in a sequence-specific manner but might provide scaffolds for co-operative binding of chromatin-associated complexes that enable spreading of Chromatin modifications.
Abstract: The role of RNA as a messenger in the expression of the genome has been long appreciated, but its functions in regulating chromatin and chromosome structure are no less interesting. Recent results have shown that small RNAs guide chromatin-modifying complexes to chromosomal regions in a sequence-specific manner to elicit transcriptional repression. However, sequence-specific targeting by means of base pairing seems to be only one mechanism by which RNA is employed for epigenetic regulation. The focus of this review is on large RNAs that act in the dosage-compensation pathways of flies and mammals. These RNAs associate with chromatin over the length of whole chromosomes and are crucial for spreading epigenetic changes in chromatin structure. They do not appear to act in a sequence-specific manner but might provide scaffolds for co-operative binding of chromatin-associated complexes that enable spreading of chromatin modifications.

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TL;DR: It is found that the observed phenotypic differences between both sisters who are cytogenetically normal, are caused by extreme skewed X‐chromosome inactivation, and suggests a genetic origin for this phenomenon.
Abstract: Fragile X syndrome is the most common form of inherited mental retardation. It is caused by the increase in length of a stretch of CGG triplet repeats within the FMR1 gene. A full mutation (> 200 repeats) leads to methylation of the CpG island and silencing of the FMR1 gene. We present here two sisters that are compound heterozygotes for a full mutation and a 53 repeat intermediate allele, one of them showing mental retardation and clinical features of an affected male (speech delay, hyperactivity, large ears, prominent jaw, gaze aversion), while the other is borderline normal (mild delay). Southern blot and FMRP expression analysis showed that the sister with mental retardation had the normal FMR1 gene totally methylated and no detectable protein, while her sister had 70% of her cells with the normal FMR1 gene unmethylated and normal FMRP levels. We found that the observed phenotypic differences between both sisters who are cytogenetically normal, are caused by extreme skewed X-chromosome inactivation. Analysis of the extended family showed that most of the other female family members that carry a pre-mutation or a full mutation showed some degree of skewing in their X-chromosome inactivation. The presence of several family members with skewed X inactivation and the direction and degree of skewing is inconsistent with a mere selection during development, and suggests a genetic origin for this phenomenon.

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TL;DR: The human X chromosome contains many genes related to cancer or to sex and reproduction, which suggest that it may play more important roles than any autosomal chromosome in the development and progression of reproductive and urologic cancers.
Abstract: In an XX female, one of the two X chromosomes has been inactivated during early embryonic life to achieve a compensation of X-linked gene products between males and females, leaving only one allele of X-linked genes functional. There are some X-linked genes escaping the X-inactivation, i.e., being expressed from both alleles. Escape from X-inactivation varies at different levels; some genes have both alleles active in some women but only one allele active in others, whereas some other genes have both alleles active in neoplastic tissue but only one allele active normally. The X-inactivation may be considered functionally equivalent to a loss of heterozygosity (LOH) for some genes, whereas escape from X-inactivation may be equivalent to functional gene amplification for others. The physiological LOH may make X-linked tumor suppressor genes lose their function more easily, compared with autosomal tumor suppressor genes, thus predisposing women to cancer formation more easily. Moreover, the human X chromosome contains many genes related to cancer or to sex and reproduction. All these properties of the X chromosome suggest that it may play more important roles than any autosomal chromosome in the development and progression of reproductive and urologic cancers.