Silencing Of X-linked Genes Research Articles

YY1 binding is a gene-intrinsic barrier to Xist-mediated gene silencing

X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. The chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as a barrier to Xist-mediated silencing until the late stages of the XCI process.

SPEN is required for Xist upregulation during initiation of X chromosome inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming.

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.

A novel approach to differentiate rat embryonic stem cells in vitro reveals a role for RNF12 in activation of X chromosome inactivation

X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.

Platypus Induced Pluripotent Stem Cells: The Unique Pluripotency Signature of a Monotreme

The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted.

Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

SummaryA hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.

Sex-specific silencing of X-linked genes by Xist RNA

X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of X(ΔTsix)Y male cells displayed ectopic Xist RNA coating compared with X(ΔTsix)X female cells. This increase reflected the inability of X(ΔTsix)Y cells to efficiently silence X-linked genes compared with X(ΔTsix)X cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in X(ΔTsix)X female cells relative to X(ΔTsix)Y male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating X(ΔTsix)Y and 39,X(ΔTsix) (X(ΔTsix)O) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because X(ΔTsix)X female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active X(ΔTsix) X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing.

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

Incomplete meiotic sex chromosome inactivation in the domestic dog.

BackgroundIn mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses.ResultsIn addition to PAR synapsis between X and Y, we observed extensive self-synapsis of part of the dog X chromosome, and rapid loss of known markers of DSB repair from that part of the X. Sequencing of RNA from purified spermatocytes and spermatids revealed establishment of MSCI. However, the self-synapsing region of the X displayed higher X-linked gene expression compared to the unsynapsed area in spermatocytes, and was post-meiotically reactivated in spermatids. In contrast, genes in the PAR, which are expected to escape MSCI, were expressed at very low levels in both spermatocytes and spermatids. Our comparative analysis was then used to identify two X-linked genes that may escape MSCI in spermatocytes, and 21 that are specifically re-activated in spermatids of human, mouse and dog.ConclusionsOur data indicate that MSCI is incomplete in the dog. This may be partially explained by extensive, but transient, self-synapsis of the X chromosome, in association with rapid completion of meiotic DSB repair. In addition, our comparative analysis identifies novel candidate male fertility genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1501-9) contains supplementary material, which is available to authorized users.

Uncoupling of X-linked gene silencing from XIST binding by DICER1 and chromatin modulation on human inactive X chromosome.

In mammals, X-inactivation process is achieved by the cis-spreading of long noncoding Xist RNA over one of the female X chromosomes. The Xist binding accumulates histones H3 methylation and H4 hypoacetylation required for X inactivation that leads to proper dosage compensation of the X-linked genes. Co-transcription of Tsix, an antisense copy of Xist, blocks the Xist coating on the Xi. In mice ES cells, an RNase III enzyme Dicer1 disrupts Xist binding and methylated H3K27me3 accumulation on the Xi. Later, multiple reports opposed these findings raising a question regarding the possible role of Dicer1 in murine X silencing. Here, we show that reduction of DICER1 in human female cells increases XIST transcripts without compromising the binding of the XIST and histone tail modifications on the Xi. Moreover, DICER1-depleted cells show differential upregulation of many human X-linked genes by binding different amounts of acetylated histone predominantly on their active promoter sites. Therefore, X-linked gene silencing, which is thought to be coupled with the accumulation of XIST and heterochromatin markers on Xi can be disrupted in DICER1 depleted human cells. These results suggest that DICER1 has no apparent effect on the recruitment of heterochromatic markers on the Xi but is required for inactivation of differentially regulated genes for themaintenance of proper dosage compensation in differentiated cells.

Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation

In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNA-mediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we describe analysis of a novel targeted mutation, Xist(INV), designed to test the function of a conserved region located in exon 1 of Xist RNA during X inactivation in mouse. We show that Xist(INV) is a strong hypomorphic allele that is appropriately regulated but compromised in its ability to silence X-linked loci in cis. Inheritance of Xist(INV) on the paternal X chromosome results in embryonic lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting Xist(INV) on the maternal X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the Xist(INV) allele. Analysis of cells that express Xist(INV) RNA demonstrates reduced association of the mutant RNA to the X chromosome, suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Two forms of XCI ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) around the four cell stage of embryonic development. In the embryonic tissues of the inner cell mass (ICM), a random form of XCI occurs in blastocysts which inactivates either the Xp or the maternal X chromosome (Xm) 1,2. Both forms of XCI require the non-coding Xist RNA which coats the inactive X chromosome (Xi) from which it is expressed. Xist plays crucial functions for the silencing of X-linked genes including Rnf12 3,4 encoding the ubiquitin ligase RLIM. Targeting a conditional knockout (KO) of Rnf12 to oocytes where RLIM accumulates to high levels, we find that the maternal transmission of the mutant X chromosome (Δm) leads to embryonic lethality due to defective imprinted XCI. We show that in Δm female embryos the initial formation of Xist clouds and Xp silencing is inhibited. In contrast, ES cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial roles to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

Active Chromatin Marks Are Retained on X Chromosomes Lacking Gene or Repeat Silencing Despite XIST/Xist Expression in Somatic Cell Hybrids

BackgroundX-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.Methodology/Principal FindingsTo assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.Conclusions/SignificanceLoss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice

In mammals, X-chromosome dosage compensation is achieved by inactivating one of the two X chromosomes in females. In mice, X inactivation is initially imprinted, with inactivation of the paternal X (Xp) chromosome occurring during preimplantation development. One theory is that the Xp is preinactivated in female embryos, because of its previous silence during meiosis in the male germ line. The extent to which the Xp is active after fertilization and the exact time of onset of X-linked gene silencing have been the subject of debate. We performed a systematic, single-cell transcriptional analysis to examine the activity of the Xp chromosome for a panel of X-linked genes throughout early preimplantation development in the mouse. Rather than being preinactivated, we found the Xp to be fully active at the time of zygotic gene activation, with silencing beginning from the 4-cell stage onward. X-inactivation patterns were, however, surprisingly diverse between genes. Some loci showed early onset (4-8-cell stage) of X inactivation, and some showed extremely late onset (postblastocyst stage), whereas others were never fully inactivated. Thus, we show that silencing of some X-chromosomal regions occurs outside of the usual time window and that escape from X inactivation can be highly lineage specific. These results reveal that imprinted X inactivation in mice is far less concerted than previously thought and highlight the epigenetic diversity underlying the dosage compensation process during early mammalian development.

Dosage Compensation in the Mouse Balances Up-Regulation and Silencing of X-Linked Genes

Dosage compensation in mammals involves silencing of one X chromosome in XX females and requires expression, in cis, of Xist RNA. The X to be inactivated is randomly chosen in cells of the inner cell mass (ICM) at the blastocyst stage of development. Embryonic stem (ES) cells derived from the ICM of female mice have two active X chromosomes, one of which is inactivated as the cells differentiate in culture, providing a powerful model system to study the dynamics of X inactivation. Using microarrays to assay expression of X-linked genes in undifferentiated female and male mouse ES cells, we detect global up-regulation of expression (1.4- to 1.6-fold) from the active X chromosomes, relative to autosomes. We show a similar up-regulation in ICM from male blastocysts grown in culture. In male ES cells, up-regulation reaches 2-fold after 2–3 weeks of differentiation, thereby balancing expression between the single X and the diploid autosomes. We show that silencing of X-linked genes in female ES cells occurs on a gene-by-gene basis throughout differentiation, with some genes inactivating early, others late, and some escaping altogether. Surprisingly, by allele-specific analysis in hybrid ES cells, we also identified a subgroup of genes that are silenced in undifferentiated cells. We propose that X-linked genes are silenced in female ES cells by spreading of Xist RNA through the X chromosome territory as the cells differentiate, with silencing times for individual genes dependent on their proximity to the Xist locus.

Rab33A: Characterization, Expression, and Suppression by Epigenetic Modification

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

A Stable Proteinaceous Structure in the Territory of Inactive X Chromosomes

Transcriptional inactivation of one copy of the X chromosome in female cells equalizes expression of X-linked genes between males and females. This "dosage compensation" is a multistep process that involves epigenetic modifications of chromatin and is induced by the expression of a large non-coding RNA termed Xist. In contrast to protein-coding mRNA molecules, which are free to diffuse and roam the entire nuclear interior, Xist is locally constrained to the territory of inactive X chromosomes by as yet unclear mechanisms. Recent results have suggested a contribution of scaffold attachment factor A (SAF-A) in the silencing of X-linked genes, maybe by inducing a local change in nuclear architecture. Here, in vivo mobility experiments demonstrate that SAF-A is a component of a highly stable proteinaceous structure in the territory of inactive X chromosomes, which might act as a platform for immobilizing Xist RNA during the maintenance phase of X inactivation.

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

X-Inactivation and Histone H4 Acetylation in Embryonic Stem Cells

In female mammalian cells, dosage compensation for X-linked genes is achieved by the transcriptional silencing, early in development, of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression ofXist,a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. We describe here the use of these cells in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, we show that an underacetylated X chromosome appears only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by Day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by Day 4 of differentiation (i.e., when deacetylation of Xi is first seen). The overall deacetylation of Xi followsXistexpression and the first appearance of a single, late-replicating X, both of which occur on Day 2. It also follows the silencing of X-linked genes. Levels of mRNA from four such genes,Hprt, G6pd, Rps4,andPgk-1,had all fallen by approximately 50% (relative to the autosomal geneAprt) by Days 2–4. The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, we note that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach we have used and that such selective deacetylation may still be part of the silencing process.

Role of late replication timing in the silencing of X-linked genes.

Cytosine methylation at promoter regions and late replication timing have both been implicated in the regulation of genes subject to X chromosome inactivation. Reported here are studies of X-linked gene replication in normal male and female cells as well as in cell hybrids that contain either a normal active X, a normal inactive X, or an inactive X chromosome that has been treated with the demethylating agent, 5-azacytidine (5aC). The relationship between replication timing and transcriptional activity was examined for XIST, XPCT, PGK1, HPRT, F9, FMR1, IDS, and G6PD, and earlier replication was generally found to be associated with increased transcriptional activity. The HPRT and G6PD genes in an untreated inactive X hybrid were among the few exceptions to this correlation in that they remain inactive, yet replicate earlier than their inactive X alleles present in normal human diploid cells. This condition of earlier replication timing may contribute to the high rates of 5aC-induced reactivation for HPRT and G6PD in this hybrid relative to other inactive X hybrids. Other anomalous cases include 5aC-induced advances in replication time for genes such as XIST and F9 whose transcription was unaltered by treatment. These and other data support a model for regulation of X-inactivated genes that involves at least two levels of control: (i) large chromosomal domains are placed into a transcriptionally nonpermissive state by late replication and (ii) transcription is blocked at the local level by promoter methylation. In addition, our observations of continued XIST expression in 5aC-treated hybrids with reactivated genes indicates that such expression is not sufficient for the maintenance of X inactivation.