Histone acetylation and X inactivation

In mammals, the levels of X-linked gene products in males and females are equalised by the silencing, early in development, of most of the genes on one of the two female X chromosomes. Once established, the silent state is stable from one cell generation to the next. In eutherian mammals, the inactive X chromosome (Xi) differs from its active homologue (Xa) in a number of ways, including increased methylation of selected CpGs, replication late in S-phase, expression of the Xistgene with binding of Xist RNA and underacetylation of core histones. The latter is a common property of genetically inactive chromatin but, in the case of Xi, it is not clear whether it is an integral part of the silencing process or simply a consequence of some other property of Xi, such as late replication. The present review describes two approaches that address this problem. The first shows that Xi in marsupial mammals also contains underacetylated H4, even though its properties differ widely from those of the eutherian Xi. The continued presence of histone underacetylation on Xi in these evolutionarily distant mammals argues for its fundamental importance. The second approach uses mouse embryonic stem cells and places H4 deacetylation in a sequence of events leading to complete X inactivation. The results argue that histone underacetylation plays a role in the stabilisation of the inactive state, rather than in its initiation. Dev. Genet. 22:65–73, 1998. © 1998 Wiley-Liss, Inc.     

Genomic and epigenomic approaches to the study of X chromosome inactivation

X chromosome inactivation represents a compelling example of chromosome-wide, long-range epigenetic gene-silencing in mammals. The cis- and trans-acting factors that establish and maintain the patterns and levels of gene expression from the active and inactive X chromosomes remain incompletely understood; however, the availability of the complete genomic sequence of the human X chromosome, together with complementary approaches that explore the computational biology, epigenetic modifications and gene expression-profiling along the chromosome, suggests that the features of the X chromosome that are responsible for its unique forms of gene regulation are increasingly amenable to experimental analysis.     

Polymorphic X-chromosome inactivation of the human TIMP1 gene.

X inactivation silences most but not all of the genes on one of the two X chromosomes in mammalian females. The human X chromosome preserves its activation status when isolated in rodent/human somatic-cell hybrids, and hybrids retaining either the active or inactive X chromosome have been used to assess the inactivation status of many X-linked genes. Surprisingly, the X-linked gene for human tissue inhibitor of metalloproteinases (TIMP1) is expressed in some but not all inactive X-containing somatic-cell hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Since many genes that escape X inactivation are clustered, we examined the expression of four genes (ARAF1, ELK1, ZNF41, and ZNF157) within approximately 100 kb of TIMP1. All four genes were expressed only from the active X chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X chromosome are specific to the TIMP1 gene. To determine if this variable inactivation of TIMP1 is a function of the hybrid-cell environment or also is observed in human cells, we developed an allele-specific assay to assess TIMP1 expression in human females. Expression of two alleles was detected in some female cells with previously demonstrated extreme skewing of X inactivation, indicating TIMP1 expression from the inactive chromosome. However, in other cells, no expression of TIMP1 was observed from the inactive X chromosome, suggesting that TIMP1 inactivation is polymorphic in human females.     

Analysis of methylation of a human X located gene which escapes X inactivation.

The gene MIC2 is located in the pseudoautosomal region at the ends of the short arms of the X and Y chromosomes. In females MIC2 escapes X inactivation. We have analyzed the methylation pattern of MIC2 on the active X, the inactive X chromosomes, and the Y chromosome. The 5' end of the gene contains a GC rich region which is unmethylated on the active X, the inactive X and on the Y. The body of the gene is characterized by variable methylation.     

Re-evaluation of the function of the male specific lethal complex in Drosophila

A set of proteins and noncoding RNAs,referred to as the male specific lethal (MSL) complex,is present on the male X chromosome in　Drosophila and has been postulated to be responsible for dosage compensation of this chromosome - the up-regulation of its expression to be　equal to that of two X chromosomes in females.This hypothesis is evaluated in view of lesser known aspects of dosage compensation such as the　fact that metafemales with three X chromosomes also have equal expression to normal females,which would require a down-regulation of each　gene copy.Moreover,when this complex is ectopically expressed in females or specifically targeted to a reporter in males,there is no increase in　expression of the genes or targets with which it is associated.These observations are not consistent with the hypothesis that the MSL complex　conditions dosage compensation.A synthesis is described that can account for these observations.     

Sexual antagonism and the evolution of X chromosome inactivation

In most female mammals, one of the two X chromosomes is inactivated early in embryogenesis. Expression of most genes on this chromosome is shut down, and the inactive state is maintained throughout life in all somatic cells. It is generally believed that X-inactivation evolved as a means of achieving equal gene expression in males and females (dosage compensation). Following degeneration of genes on the Y chromosome, gene expression on X chromosomes in males and females is upregulated. This results in closer to optimal gene expression in males, but deleterious overexpression in females. In response, selection is proposed to favor inactivation of one of the X chromosomes in females, restoring optimal gene expression. Here, we make a first attempt at shedding light on this intricate process from a population genetic perspective, elucidating the sexually antagonistic selective forces involved. We derive conditions for the process to work and analyze evolutionary stability of the system. The implications of our results are discussed in the light of empirical findings and a recently proposed alternative hypothesis for the evolution of X-inactivation.     

Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation

Nuclear transfer (NT) studies have provided insight into the functional importance of epigenetic alteration of the X chromosomes during X-inactivation. Uniparental embryos created by NT have been informative as to the time and location at which the imprint controlling extraembryonic X-inactivation is established. Experiments with female somatic cells, have demonstrated that the inactive X chromosome (Xi) is reactivated after NT, leading to random X-inactivation in the embryonic lineages of cloned embryos. However, in the extraembryonic lineages of clones, epigenetic information from the donor cell nucleus persists, leading to preferential inactivation of the donor cell's inactive X in the placenta of cloned animals. These results suggest epigenetic information established during embryonic X-inactivation is functionally equivalent to the gametic imprint.     

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.     

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.     

Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands

The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.     

Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts

X chromosome inactivation (XCI) is an essential epigenetic process that ensures X‐linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.     

Role of the male specific lethal (msl) genes in modifying the effects of sex chromosomal dosage in Drosophila

Immunostaining of chromosomes shows that the male-specific lethal (MSL) proteins are associated with all female chromosomes at a low level but are sequestered to the X chromosome in males. Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the X and lower on the autosomes in males than in females. However, the staining pattern of acetylation and the mof gene product, a putative histone acetylase, in msl mutant males returns to a uniform genome-wide distribution as found in females. Gene expression on the autosomes correlates with the level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are maintained with either an increase or decrease of acetylation, suggesting that the MSL complex renders gene activity unresponsive to H4Lys16 acetylation. Evidence was also found for the presence of nucleation sites for association of the MSL proteins with the X chromosome rather than individual gene binding sequences. We suggest that sequestration of the MSL proteins occurs in males to nullify on the autosomes and maintain on the X, an inverse effect produced by negatively acting dosage-dependent regulatory genes as a consequence of the evolution of the X/Y sex chromosomal system.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.