X chromosome inactivation in nuclear transfer ES cells

Nuclear transfer ES (ntES) cells are established from cloned blastocysts generated by somatic cell nuclear transfer and are expected to be an important resource for regenerative medicine. However, cloned mammals, generated by similar methods, show various abnormalities, which suggest disordered gene regulation. Random X chromosome inactivation (XCI) has been observed to take place in cloned female mouse embryos, but XCI does not necessarily occur according to Xce strength, a genetic element that determines the likelihood of each X chromosome to be inactivated. This observation suggests incomplete reprogramming of epigenetic marks related to XCI. Here, we investigated XCI in ntES cell lines, which were established using differentiated embryoid bodies that originated from a female mouse ES cell line. We examined Xist RNA localization, histone modifications in the Xist locus, and XCI choice. We did not find substantial differences between the ntES lines and their parental ES line. This suggests that the Xist locus and the epigenetic marks involved in XCI are reprogrammed by nuclear transfer and subsequent ntES cell establishment. In contrast to skewed XCI in cloned mice, our observations indicate that normal XCI choice takes place in ntES cells, which supports the goal of safe therapeutic cloning for clinical use.     

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.     

The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3' to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3' to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.     

X chromosome reactivation and regulation in cloned embryos

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.     

Antagonistic interactions between the cAMP-dependent protein kinase and Tor signaling pathways modulate cell growth in Saccharomyces cerevisiae

In mammals, X-chromosome inactivation (XCI) equalizes X-linked gene expression between XY males and XX females and is controlled by a specialized region known as the X-inactivation center (Xic). The Xic harbors two chromatin interaction domains, one centered around the noncoding Xist gene and the other around the antisense Tsix counterpart. Previous work demonstrated the existence of a chromatin transitional zone between the two domains. Here, we investigate the region and discover a conserved element, RS14, that presents a strong binding site for Ctcf protein. RS14 possesses an insulatory function suggestive of a boundary element and is crucial for cell differentiation and growth. Knocking out RS14 results in compromised Xist induction and aberrant XCI in female cells. These data demonstrate that a junction element between Tsix and Xist contributes to the initiation of XCI.     

Expression of Xist RNA is sufficient to initiate macrochromatin body formation

MacroH2A1 is a histone variant that is found as a component of the inactive X chromosome where it is detected as a dense accumulation called a macrochromatin body (MCB). Macrochromatin bodies co-localize with Xist RNA, which is an untranslated RNA that is expressed exclusively from the inactive X chromosome of placental mammals. However, no studies to date have investigated whether Xist RNA expression is necessary or sufficient to cause the formation of MCBs. Here we show that expression of Xist RNA is sufficient to cause the formation of MCBs even when Xist is expressed from an inducible transgene at ectopic autosomal sites. Macrochromatin bodies form at sites of transgenic Xist expression in differentiating mouse ES cell lines and transgenic fibroblasts, but MCBs cannot form in undifferentiated ES cells even after prolonged Xist expression. The kinetics of MCB formation revealed that Xist expression precedes MCB formation and that differentiating ES cells undergo a rapid and synchronous transition that renders them competent to form MCBs. Once MCBs have formed, continued expression of Xist is required for their maintenance. These results show that Xist RNA and macroH2A1 function in a common pathway. Expression of Xist in a permissive nuclear environment is sufficient to initiate a chromatin-remodeling event culminating in the incorporation of macroH2A1. The results also strongly suggest the existence of additional regulatory factors for X inactivation that are regulated developmentally. In addition, we present evidence that macroH2A1 density is not simply a measure of the general degree of DNA compaction.     

Xist-mediated chromatin changes that establish silencing of an entire X chromosome in mammals

X chromosome inactivation (XCI) ensures an equal gene dosage between the sexes in placental mammals. Xist, a modular multi-domain X-encoded long non-coding RNA coats the X chromosome in cis during XCI. Xist recruits chromatin remodelers and repressor complexes ensuring silencing of the inactive X (Xi). Here, we review the recent work focused on the role of Xist functional repeats and interacting RNA-binding factors in the establishment of the silent state. Xist orchestrates recruitment of remodelers and repressors that first facilitate removal of the active chromatin landscape and subsequently direct the transition into a repressive heterochromatic environment. Some of these factors affect silencing on a chromosome-wide scale, while others display gene-specific silencing defects. The temporal order of recruitment shows each silencing step is party dependent on one another. After the Xi is established, many of the factors are dispensable, and a different repertoire of proteins ensure the silenced Xi is maintained and propagated.