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X inactivation Xplained Anton Wutz1 and Joost Gribnau2 Random inactivation of one of the two female X chromosomes establishes dosage compensation between XY males and XX females in placental mammals. X inactivation is controlled by the X inactivation center (Xic). Recent advances in genome sequencing show that the Xic has evolved from an ancestral vertebrate gene cluster in placental mammals and has undergone separate rearrangements in marsupials. The Xic ensures that all but one X chromosome per diploid genome are inactivated. Which chromosome remains active is randomly chosen. Pairing of Xic loci on the two X chromosomes and alternate states of the X chromosomes before inactivation have recently been implicated in the mechanism of random choice. Chromosome-wide silencing is then initiated by the noncoding Xist RNA, which evolved with the mammalian Xic and covers the inactive X chromosome. Addresses 1 Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria 2 Department of Reproduction and Development, Erasmus University Medical Center, Rotterdam, The Netherlands Corresponding author: Wutz, Anton ([email protected]) and Gribnau, Joost ([email protected]) Current Opinion in Genetics & Development 2007, 17:387–393 This review comes from a themed issue on Differentiation and gene regulation Edited by Denis Duboule and Frank Grosveld Available online 14th September 2007 0959-437X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2007.08.001 Introduction Mammals have evolved a complex set of functions for gene regulation allowing elegant control of expression during development. One of the most intriguing forms of gene regulation leads to exclusive gene expression from one single parental allele. Different mechanisms are used to achieve monoallelic gene expression ranging from parental imprinting [1] to odorant receptor choice in the olfactory system [2]. These processes are tailored to suit the requirements of different biological systems. For instance, the immunoglobulin heavy chain rearrangements in B-lymphopoiesis generate a pool of cells each expressing one specific antibody, which allows for clonal selection in the immune system [3]. This process is random with respect to the parental origin of the locus. In contrast, imprinted genes show exclusive expression from one parental allele [4]. In mammals dosage www.sciencedirect.com compensation of X-linked genes between the sexes is established by inactivation of one of the two female Xs (reviewed in [5]). In mice X chromosome inactivation (XCI) is random in the embryo, and imprinted in extraembryonic tissues, where the paternally inherited X chromosome is inactivated [6]. X inactivation, thus, exemplifies the functions of the mammalian cell nucleus in domain regulation and monoallelic expression. Here, we review recent progress in understanding of the evolution of the mammalian dosage compensation system, the mechanism of random choice and chromosome-wide inactivation. Evolution does dosage compensation Dosage compensation systems adjust the gene doses of sex chromosome linked genes between the sexes and are observed throughout the animal kingdom. Different strategies to accomplish dosage compensation are used by fruit flies, worms, and mammals (reviewed in [7]). Interestingly, no evidence for efficient dosage compensation has been found by genome-wide expression analysis in chicken and finches [8]. Differences in Z gene dosis between ZZ males and ZW females result in elevated Z gene levels in males and might contribute to sexual differentiation in birds. The observation of dosis compensation systems in other organisms must therefore be explained by an additional advantage rather than an absolute necessity during evolution. This hypothesis is highlighted by the observation that many genes escape XCI in human [9], and supported by the intriguing finding that monoallelic expression of the entire X chromosome has facilitated the evolution of trichromatic color vision in monkeys [10]. The mammalian dosage compensation system is controlled to a large extent by the X inactivation center (Xic) located on the X chromosome. The Xic regulates counting of the number of X chromosomes and contains the noncoding Xist RNA gene, which localizes specifically to the inactive X chromosome (Xi) and triggers chromosome-wide gene repression. Recent findings show that this locus has apparently evolved exclusively in placental mammals [11,12,13]. In marsupials and the more distant vertebrates, such as frog or fish, the genomic region around the Xic has an entirely different structure with evidence for multiple rearrangements (Figure 1). Interestingly, a different study indicates that the Xist gene has newly emerged from an ancestral protein-coding gene Lnx3 during mammalian evolution [14], indicating that XCI in marsupials requires an alternative mechanism for dosage compensation. Current Opinion in Genetics & Development 2007, 17:387–393 388 Differentiation and gene regulation Figure 1 Evolution of the X inactivation center. (a) A scheme of the Xic locus in placental mammals is shown. The Xist and regulatory Tsix transcript are indicated. Genes that are derived from vertebrate protein-coding genes and have lost protein-coding potential in eutheria are indicated in blue. Boxed genes serve as anchors for identifying the genomic region in vertebrates and marsupials. (b) The ancestral vertebrate gene cluster corresponding to the mammalian Xic region has undergone an expansion during placental mammal evolution. The protein-coding LNX3 gene shown in green shares sequence homology in a few exons with the noncoding Xist and is believed to be the ancestral Xist gene. (c) In the marsupial lineage the ancestral vertebrate gene cluster has been rearranged and split in two clusters of genes, which map to distant regions on the X. Xist has exclusively evolved in placental mammals and in marsupial genomes the LNX3 protein-coding gene is found. Regulation of randomness How random X inactivation is established is unclear at present. Recent progress in the search for mechanisms driving the XCI counting and choice process, which mark the initiation of XCI, has produced evidence that multiple regulatory systems may be involved. Different observations made with aneuploid, and tetraploid cells indicated that one X chromosome remains active per diploid genome after completion of XCI. These and other findings led to the hypothesis that counting and choice in XCI can be explained by the action of an autosomal blocking factor (BF), which protects one X chromosome per diploid genome from inactivation (Figure 2a). Studies aimed to identify the blocking factor binding site have resulted in the identification of a 1.2 kb candidate element, DXPas34, which is located 30 of Xist [15]. Deletion of this element resulted in ectopic XCI in male cells, indicating a role for this element in the counting process [16]. Surprisingly, a different study describing a similar deletion of DXPas34 reported no ectopic XCI in Current Opinion in Genetics & Development 2007, 17:387–393 male cells, indicating that more work has to be done to clarify this topic [17]. The real nature of the blocking factor remains elusive so far, and could be a nuclear episome, or a limited protein or protein complex. Based on the blocking factor model using theoretical considerations a symmetry-breaking model has been proposed, in which many diffusible molecules are quantitatively sequestered on the active X chromosome by a mechanism that involves intermolecular BF interactions [18] (Figure 2b). Close proximity of the Xic loci would benefit this model to allow binding equilibration to occur fast enough to be able to discriminate between an active blocked and an inactive nonblocked X chromosome. Indeed, trans-interaction of X chromosomes by Xic–Xic pairing has been observed [19,20]. Based on this observation a mutual exclusive choice model has been proposed that posits coordinate trans-regulation of the interacting Xics [21] (Figure 2d). Trans-vection is known from the fruit fly [22] and may aid www.sciencedirect.com X inactivation Xplained Wutz and Gribnau 389 Figure 2 Different models for the initiation of random XCI. (a) An autosomally encoded blocking factor (BF) protects one X chromosome from inactivation. (b) Breaking of the symmetry in particle binding leads to accumulation of many diffusible molecules on one X chromosome protecting it from inactivation. (c) Alternate epigenetic states mark the future Xa and Xi before the initiation of XCI. (d) Both X chromosomes meet in space and determine the future Xa and Xi. (e) In a stochastic model each X chromosome has a certain probability to be inactivated. blocking factor binding. The precise function of the Xic interaction remains to be determined. Notably, XCI counting is not affected in female cells with a 65 kb deletion 30 of Xist which shows a loss of Xic pairing [19]. In several models, the XCI counting and choice process is anticipated to be triggered by developmental or differentiation cues. Nevertheless, X chromosomes might be distinguished before inactivation by assuming alternative states (Figure 2c). Recently, it has been reported that cohesion of sister chromatids is regulated differentially www.sciencedirect.com between the two female X chromosomes [23]. This could highlight an inherent difference between the two genetically identical chromosomes in at least part of the cell population. The different states may represent blocking factor binding to one chromosome, or accessibility or transcriptional differences between the two Xics, although more work is required to establish the molecular basis of this phenomenon. The potential risk of female lethality dictates a need for a precise counting process. This assumption has spurred a Current Opinion in Genetics & Development 2007, 17:387–393 390 Differentiation and gene regulation hunt for regulatory mechanisms. Nevertheless, any given mechanism of XCI counting and choice requires a stochastic element. In principle, this could be achieved simply by inactivating each X chromosome with a certain fixed and fine-tuned probability. It has been shown that chaotic choice results if Tsix is disrupted on both Xic alleles [24]. Surprisingly, this can result in viable female mice, albeit with a low frequency [24] (Figure 2e). Therefore, chaotic choice may represent the basic mechanism underlying the XCI counting and choice process. Currently, the molecular identity for factors involved in the counting process remains unknown [5]. Suggestions that beside an autosomally encoded blocking factor an X-linked competence factor might stimulate X inactivation further complicate the scenarios [21]. The molecular underpinning is clearly complex and it is hard to see how to discriminate between different models if not by analysis of molecular components. XCI starts with the accumulation of Xist along the future inactive X chromosome. Recent work indicates that Xist up-regulation is the consequence of sex-specific induction of Xist transcription on the future Xi and not stabilization of the Xist RNA [25]. In differentiated cells Xist repression on the Xa is mediated by DNA methylation [26]. A recent study shows that the methyl-DNA binding protein Mbd2 is required for Xist repression and, thus, provides a link to repressor complexes [27]. During the early stages of XCI, Xist expression is regulated by the Tsix gene. Tsix is a long untranslated RNA, which acts predominantly in the nucleus and is transcribed in antisense direction over the Xist gene. A recent report shows that Tsix does not require splicing for its function [28], leaving open the question whether the act of transcription or the Tsix RNA itself are required for Xist repression. Tsix function requires the intronic DXPas34 element [16,17]. Deletion of this element results in nonrandom XCI of the mutated X chromosome and a defect in imprinted XCI [17]. DXPas34 harbors many putative CTCF binding sites, frequently flanked by putative YY1 sites [29]. Several of these CTCF sites show differential methylation in gametes and somatic cells, implying a direct role for this element in imprinted and random XCI [30]. Moreover, analysis of YY1 mutant embryos has indicated a role for this protein in the regulation of Tsix and Xist [29], but it remains to be established if YY1 and CTCF act as bona fide blocking factors, or are required to perform structural roles for Xic regulation. A conundrum is the lack of conservation of Tsix and DXPas34 between the mouse and human XIC. It has been observed in human and bovine cells that the antisense TSIX RNA is coexpressed with XIST and, thus, apparently does not act to repress XIST in all mammals [31,32]. This raises the possibility that evolution has selected different molecular components for regulating randomness in different mammals. Current Opinion in Genetics & Development 2007, 17:387–393 Building a cis-limited repressive nuclear compartment The human Xi is comprised of a heterogeneous chromatin landscape [33]. Not all genes are repressed on the Xi and a relatively large set of genes escape from inactivation in human. Recently, a gene activity profile has been derived for the human Xi [9]. The availability of the human X chromosome sequence [34] allowed the identification of X enriched sequence elements [35,36]. Importantly, a prediction for gene inactivation and escape of inactivation was reported [35]. The identified sequences originate in part from genomic repeat elements of the L1 long interspersed elements (LINE) class, which were already implicated in spreading of X inactivation [37]. Experimental support for this hypothesis comes from analyses of mice carrying the T(X;4)37H X;autosome translocation chromosome. This study shows that spreading of Xist RNA is attenuated at the autosomal boundary of the translocation, which has a low abundance of LINE elements [38]. Albeit L1 elements are not exclusive to the X chromosome, they might be part of the features on the X chromosome that facilitate spreading of the dosage compensation complex. Recent studies indicate that in the interphase nucleus the human Xi is organized into an outer rim of genes and a repeat-rich core, to which Xist localizes [39]. In differentiated mouse ES cells, Xist forms a repressive compartment from which the transcription machinery is excluded as a first step in X inactivation [40]. This compartment initially does not contain genes and is also not sufficient for gene silencing [40]. Genes become repressed and associate with the repressive compartment upon silencing, which requires the repeat-A sequences on the 50 -end of Xist RNA [41] (Figure 3a). How silencing is established and how it is restricted to one specific chromosome requires further studies. The function of Xist in gene silencing has been reported to be restricted to early development and specific somatic cell types such as precursors in the blood system [42] (Figure 3b). This suggests that the pathways for gene silencing are regulated in a cell type-specific manner. Once X inactivation has been established the silent X chromosome is stably maintained in somatic cells. Chromosome-wide chromatin analysis of X-linked epigenetic modifications confirmed many cytological observations, including hypoacetylation of histone H4, accumulation of histone H3, K9, and K27 tri-methylation and accumulation of histone variant macroH2A on the Xi [43,44] (Figure 3b). These results suggest that a wide variety of chromatin modifiers including Polycomb group complexes, histone deacetylases, and DNA methylases act in concert to maintain repression of the Xi [45]. Beside profiles of histone modification marks along the X chromosome, a DNA methylation profile of the X chromosome has also been established [46]. This study shows that in www.sciencedirect.com X inactivation Xplained Wutz and Gribnau 391 Figure 3 Silencing of one X chromosome by Xist. (a) The two X chromosome territories are depicted. Xist RNA produced from the Xic locus accumulates on the future Xi. This leads to the formation of a nuclear compartment that is devoid of the transcription machinery, such as RNA polymerase II (Pol II). Polycomb group complexes are recruited to the Xist-covered chromosome and establish chromosome-wide histone modifications. The genes localize to the outside this compartment and are silenced in a manner that requires the Xist repeat-A sequence. Silencing of genes coincides with their association with the Xist compartment. (b) A summary of chromatin modifications during initiation of X inactivation. Xist RNA accumulation is followed by recruitment of Polycomb repressor complex 1 (PRC1), which mediates histone H2A lysine 119 ubiquitinylation (H2AK119ub1), and PRC2, which mediates histone H3 lysine 27 tri-methylation (H3K27me3). Accumulation of histone H4 lysine 20 monomethylation can also be observed on the X. In this phase Xist triggers gene silencing. Initially X inactivation is reversible and genes become reactivated if Xist is lost. If cell differentiation progresses histone macroH2A becomes enriched on the Xi and histone H4 is hypoacetylated. Later during the XCI process DNA cytosine methylation marks the promoters of genes on the Xi. In this phase X inactivation is irreversible and Xist is not required for maintenance of the silent state. In somatic cells Xist does not have the capacity to initiate chromosome-wide silencing. Thus, the silencing function of Xist depends on the Xist repeat-A sequence and a defined cellular context. contrast to CpG island methylation found on the Xi, DNA methylation is surprisingly more abundant on the Xa and appears to be confined to gene body sequences on the Xa. This finding may be related to the doubling of gene expression of the single Xa relative to autosomes [47]. Intriguingly, in female XX ES cells global DNA hypomethylation is observed [48]. Could this possibly indicate a potential function of demethylation in the counting process? The same epigenetic marks appear to be used at different places for different regulatory functions in X inactivation. This highlights a crucial role for spatial organization in complex gene regulatory mechanisms. The Barr body has been originally observed to locate sometimes close to the nucleolus [27]. A recent investigation has quantified the location of the Xi in differentiating ES cells and demonstrated that the association of the Xi with the nucleolus occurs in S phase [49]. This has led to www.sciencedirect.com speculation about a role for the subnuclear localization in providing an environment for stable replication of heterochromatin. It has been shown before that the Xi replicates out of synchrony with other chromosomes either early in S-phase in preimplantation embryos and late in S-phase in embryonic cells after implantation [6]. This highlights a spatial and temporal separation of the facultative heterochromatin of the Xi. Deletion of the Xist gene has been shown to result in loss of perinucleolar position of the Xi, but reactivation of the chromosome is limited to single genes in occasional cells [49]. Moreover late replication and chromosome-wide histone H4 hypoacetylation are maintained independent of Xist, yet, histone methylation marks and macroH2A are lost without Xist [50–52]. These results indicate that many superimposed epigenetic mechanisms maintain silencing of the Xi, and the exact role of the temporal association of the Xi with the nucleolus in maintaining the silent state remains to be determined. Current Opinion in Genetics & Development 2007, 17:387–393 392 Differentiation and gene regulation Conclusion and outlook There is increasing evidence that indexing of the information stored in the genome is aided by chromatin modifications such as histone methylation and acetylation. A combination of these signals has been proposed to constitute a histone code [53]. Recently, it has become clear that this is only one side of the full story and that nuclear positioning and compartmentalization carry an equal if not greater weight [54]. The chromosome-wide phenomenon of X inactivation might hold promise to further unravel the interplay between chromatin modifications and compartmentalization and will shed light on the gene regulatory repertoire of mammals. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Yang PK, Kuroda MI: Noncoding RNAs and intranuclear positioning in monoallelic gene expression. Cell 2007, 128:777-786. 2. Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R: Interchromosomal interactions and olfactory receptor choice. Cell 2006, 126:403-413. 3. Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, Busslinger M: Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev 2004, 18:411-422. 4. Pauler FM, Koerner MV, Barlow DP: Silencing by imprinted noncoding RNAs: is transcription the answer? Trends Genet 2007, 23:284-292. 5. Heard E, Disteche CM: Dosage compensation in mammals: fine-tuning the expression of the X chromosome. Genes Dev 2006, 20:1848-1867. 6. Takagi N, Sasaki M: Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 1975, 256:640-642. 7. Lucchesi JC, Kelly WG, Panning B: Chromatin remodeling in dosage compensation. Annu Rev Genet 2005, 39:615-651. 8. Itoh Y, Melamed E, Yang X, Kampf K, Wang S, Yehya N, Van Nas A, Replogle K, Band MR, Clayton DF et al.: Dosage compensation is less effective in birds than in mammals. J Biol 2007, 6:2. 9. Carrel L, Willard HF: X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature 2005, 434:400-404. This paper contains a comprehensive X inactivation profile of the human X chromosome in fibroblast-like cells. About 15% of X-linked genes escape inactivation to some degree. Variable patterns of escaping genes are found between different cell lines suggesting a potential expression heterogeneity among females. 10. Jacobs GH, Williams GA, Cahill H, Nathans J: Emergence of novel color vision in mice engineered to express a human cone photopigment. Science 2007, 315:1723-1725. 11. Davidow LS, Breen M, Duke SE, Samollow PB, McCarrey JR, Lee JT: The search for a marsupial XIC reveals a break with vertebrate synteny. Chromosome Res 2007, 15:137-146. In this report, the authors fail to identify an ortholog of Xist in the marsupial Monodelphis domestica. Cytological mapping of the area homologs to the eutherian XIC indicates that this area is rearranged in marsupials. 12. Hore TA, Koina E, Wakefield MJ, Marshall Graves JA: The region homologous to the X-chromosome inactivation centre has been disrupted in marsupial and monotreme mammals. Chromosome Res 2007, 15:147-161. Current Opinion in Genetics & Development 2007, 17:387–393 This study describes the localization of the genes flanking the XIST gene in eutherians along the marsupial X chromosome. The authors report that XIST is absent in marsupials and monotremes and originated in a genomic region that expanded in early mammals. The region homologous to the eutherian XIC was disrupted in marsupials and monotreme lineages. 13. Shevchenko AI, Zakharova IS, Elisaphenko EA, Kolesnikov NN, Whitehead S, Bird C, Ross M, Weidman JR, Jirtle RL, Karamysheva TV et al.: Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials. Chromosome Res 2007, 15:127-136. The localization of the sequence interval between the eutherian genes flanking XIST is analyzed in two different marsupial species Monodelphis domestica and Didelphis virginia. The study indicates a breakpoint in this region in both marsupial species. 14. Duret L, Chureau C, Samain S, Weissenbach J, Avner P: The Xist RNA gene evolved in eutherians by pseudogenization of a protein-coding gene. Science 2006, 312:1653-1655. Sequence analysis of XIST and flanking regions in different eutherian species and in opposum, chicken and frog reveals that XIST evolved at least in part from an ancestral protein-coding gene Lnx3. This study indicates that XIST is absent in marsupials suggesting the presence of different mechanisms of XCI in eutherians and marsupials. 15. Debrand E, Chureau C, Arnaud D, Avner P, Heard E: Functional analysis of the DXPas34 locus, a 30 regulator of Xist expression. Mol Cell Biol 1999, 19:8513-8525. 16. Vigneau S, Augui S, Navarro P, Avner P, Clerc P: An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation. Proc Natl Acad Sci USA 2006, 103:7390-7395. 17. Cohen DE, Davidow LS, Erwin JA, Xu N, Warshawsky D, Lee JT: The DXPas34 repeat regulates random and imprinted X inactivation. Dev Cell 2007, 12:57-71. 18. Nicodemi M, Prisco A: Symmetry-breaking model for X-chromosome inactivation. Phys Rev Lett 2007, 98:108104. 19. Bacher CP, Guggiari M, Brors B, Augui S, Clerc P, Avner P, Eils R, Heard E: Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat Cell Biol 2006, 8:293-299. This study and the study by Xu et al. [20] indicate that both Xics transiently pair at the onset of XCI in female cells. Analysis of Xic–Xic pairing in Tsix mutant cell lines and cell lines with single copy and multicopy Xic transgenes indicates that colocalization may be necessary but not sufficient for correct counting and choice. 20. Xu N, Tsai CL, Lee JT: Transient homologous chromosome pairing marks the onset of X inactivation. Science 2006, 311:1149-1152. Similar to Bacher et al. [19] Xic pairing is reported during the early stages of XCI. Also in this study Tsix and Xite mutant cell lines and cell lines with autosomal Xic transgenes have been analyzed with respect to Xic pairing. Results with Xite and Tsix mutant cell lines indicate that these sequences are required for Xic–Xic pairing. Analysis of cell lines with small Tsix or Xite fragments capable of inducing Xic-autosome pairing, indicates that Xic pairing is an intrinsic part of the counting and choice process. 21. Anguera MC, Sun BK, Xu N, Lee JT: X-chromosome kiss and tell: how the xs go their separate ways. 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Nat Genet 2002, 32:195-200. www.sciencedirect.com X inactivation Xplained Wutz and Gribnau 393 25. Sun BK, Deaton AM, Lee JT: A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol Cell 2006, 21:617-628. 26. Beard C, Li E, Jaenisch R: Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev 1995, 9:2325-2334. 27. Barr H, Hermann A, Berger J, Tsai HH, Adie K, Prokhortchouk A, Hendrich B, Bird A: Mbd2 contributes to DNA methylationdirected repression of the xist gene. Mol Cell Biol 2007, 27:3750-3757. 28. Sado T, Hoki Y, Sasaki H: Tsix defective in splicing is competent to establish Xist silencing. Development 2006, 133:4925-4931. 29. Donohoe ME, Zhang LF, Xu N, Shi Y, Lee JT: Identification of a Ctcf cofactor, Yy1, for the X chromosome binary switch. Mol Cell 2007, 25:43-56. 30. Prissette M, El-Maarri O, Arnaud D, Walter J, Avner P: Methylation profiles of DXPas34 during the onset of X-inactivation. Hum Mol Genet 2001, 10:31-38. 31. Migeon BR, Lee CH, Chowdhury AK, Carpenter H: Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation. Am J Hum Genet 2002, 71:286-293. 32. Farazmand A, Basrur PK, Stranzinger G, Graphodatskaya D, Reyes ER, King WA: Expression of XIST sense and antisense in bovine fetal organs and cell cultures. Chromosome Res 2004, 12:275-283. 33. Chadwick BP, Willard HF: Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome. Proc Natl Acad Sci USA 2004, 101:17450-17455. 34. Ross MT, Grafham DV, Coffey AJ, Scherer S, McLay K, Muzny D, Platzer M, Howell GR, Burrows C, Bird CP et al.: The DNA sequence of the human X chromosome. Nature 2005, 434:325-337. 35. Carrel L, Park C, Tyekucheva S, Dunn J, Chiaromonte F, Makova KD: Genomic environment predicts expression patterns on the human inactive X chromosome. PLoS Genet 2006, 2:e151. 40. 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Brinkman AB, Roelofsen T, Pennings SW, Martens JH, Jenuwein T, Stunnenberg HG: Histone modification patterns associated with the human X chromosome. EMBO Rep 2006, 7:628-634. 44. Valley CM, Pertz LM, Balakumaran BS, Willard HF: Chromosomewide, allele-specific analysis of the histone code on the human X chromosome. Hum Mol Genet 2006, 15:2335-2347. 45. Masui O, Heard E: RNA and protein actors in x-chromosome inactivation. Cold Spring Harb Symp Quant Biol 2006, 71:419-428. 46. Hellman A, Chess A: Gene body-specific methylation on the active X chromosome. Science 2007, 315:1141-1143. 47. Nguyen DK, Disteche CM: Dosage compensation of the active X chromosome in mammals. Nat Genet 2006, 38:47-53. 48. Zvetkova I, Apedaile A, Ramsahoye B, Mermoud JE, Crompton LA, John R, Feil R, Brockdorff N: Global hypomethylation of the genome in XX embryonic stem cells. Nat Genet 2005, 37:1274-1279. 49. 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Current Opinion in Genetics & Development 2007, 17:387–393