Barring gene expression after XIST: maintaining facultative heterochromatin on the inactive X

X chromosome inactivation refers to the developmentally regulated process of silencing gene expression from all but one X chromosome per cell in female mammals in order to equalize the levels of X chromosome derived gene expression between the sexes. While much attention has focused on the genetic and epigenetic events early in development that initiate the inactivation process, it is also important to understand the events that ensure maintenance of the inactive state through subsequent cell divisions. Gene silencing at the inactive X chromosome is irreversible in somatic cells and is achieved through the formation of facultative heterochromatin (visible as the Barr body) that is remarkably stable and faithfully preserved. Here we review the many features of inactive X chromatin in terminally differentiated cells and address the highly redundant mechanisms of maintaining the inactive X chromatin.     

Repressive but not activating epigenetic modifications are aberrant on the inactive X chromosome in live cloned cattle

X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones. In contrast, H3K4me3, an active gene marker, is obviously missing from the inactive X chromosome in all cattle studied. This suggests that the disappearance of active histone modifications (H3K4me3) seems to be more important for X inactivation than deposition of marks associated with heterochromatin (DNA methylation, H3K27me3 and H3K9me2). It also implies that even apparently normal clones may have subtle abnormalities in repressive, but not activating epigenetic modifications on the inactive X when they survive to term. We also found that the histone H3 methylations were enriched and co-localized at q21-31 of the active X chromosome, which may be associated with an abundance of LINE1 repeat elements.     

SETting the stage. Eed-Enx1 leaves an epigenetic signature on the inactive X chromosome

Despite evidence implicating the Polycomb group protein, Eed (embryonic ectoderm development protein) in imprinted X inactivation, a similar role in random X inactivation in the embryo has remained an open question. Brockdorff and colleagues now report that Eed, along with its binding partner Enx1, transiently associates with the inactive X chromosome (Xi) and likely contributes to the epigenetic signature and long-term stability of the Xi heterochromatin.     

Insect sex chromosomes

A presumptive mechanism of X inactivation has been investigated by using tritiated uridine-induced chromosome aberrations to distinguish active from inactive X chromosome arms in the insect Gryllotalpa fossor. Previous work on therian mammals has shown that constitutive and facultative heterochromatin are less susceptible to breakage by ³H-Urd than euchromatin (active). The present study indicates that, irrespective of the presence of two X chromosomes in females, only one of these is affected as in males and that the total number of aberrations induced by ³H-Urd in both male and female Gryllotalpa is the same. This suggests that in the female only one arm of one X chromosome is active and that a facultative heterochromatinization of the homologous arm of the other X is operative coupled with the presence of constitutive heterochromatin in the second arm of both X chromosomes.     

X-chromosome inactivation in development and cancer

X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development. Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combination of chromatin associated proteins, DNA methylation and nuclear organization. However, sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells and disappearance of the Barr body is frequently observed in cancer cells. In this review we summarise current knowledge on the epigenetic changes that accompany X inactivation and discuss the extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast cancer.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Structural heterochromatin and X-chromosome inactivation]

Our previous studies on the expression of the G6PD and alpha-GAL genes from the X chromosome of inter-specific hybrids of voles of the Microtus genus have demonstrated an unusual pattern of X-inactivation in the parents. The observed phenomenon was explained as the presumable result of nonrandom inactivation of the X chromosomes with a heterochromatin block in crosses involving Microtus arvalis whose X lacks a heterochromatin region and also of random X inactivation when both parents had heterochromatin blocks on the Xs. Based on known models, we discuss here the possible mechanisms of the effect of heterochromatin on X-inactivation; we give preference to the model postulating binding of nonhistone protein to the inactivation centre as the key event. The hypothesis we offer suggests change in chromatin conformation in the inactivation centre during packaging of heterochromatic region of a chromosome; the protein molecules diffusing along the chromosome towards the heterochromatin region by the "facilitated diffusion" mechanism may happen to be in the region of the X-inactivation centre, which, being in a favorable state, binds specifically to it; as a consequence, the binding probability of protein to heterochromatin increases as compared to chromosome without heterochromatin block.     

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.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Misbehaviour of XIST RNA in Breast Cancer Cells

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors.     

The WSTF-ISWI Chromatin Remodeling Complex Transiently Associates with the Human Inactive X Chromosome during Late S-Phase Prior to BRCA1 and γ-H2AX

Replicating the genome prior to each somatic cell division not only requires precise duplication of the genetic information, but also accurately reestablishing the epigenetic signatures that instruct how the genetic material is to be interpreted in the daughter cells. The mammalian inactive X chromosome (Xi), which is faithfully inherited in a silent state in each daughter cell, provides an excellent model of epigenetic regulation. While much is known about the early stages of X chromosome inactivation, much less is understood with regards to retaining the Xi chromatin through somatic cell division. Here we report that the WSTF-ISWI chromatin remodeling complex (WICH) associates with the Xi during late S-phase as the Xi DNA is replicated. Elevated levels of WICH at the Xi is restricted to late S-phase and appears before BRCA1 and γ-H2A.X. The sequential appearance of WICH and BRCA1/γ-H2A.X implicate each as performing important but distinct roles in the maturation and maintenance of heterochromatin at the Xi.