Chromatin reprogramming of male somatic cell-derived <i>Xist</i> and <i>Tsix</i> in ES hybrid cells

Abstract

In mammalian somatic cells, the X chromosome is active in XY males, whereas one X chromosome is inactivated in XX females. On the active male X chromosome, the Xist and Tsix genes are transcribed in undifferentiated cells of pre-implantation embryos (undifferentiated state) and then down-regulated upon cell differentiation (differentiated state). To explore the epigenetic mechanism involved in the on-off switching of Xist and Tsix transcription in the active X chromosome, male somatic cells were hybridized with male embryonic stem (ES) cells. Fluorescence in situ hybridization analysis revealed that the Xist gene derived from somatic cells was derepressed, as shown by the advent of two pinpoint signals. This was confirmed by strand-specific RT-PCR of Xist and Tsix genes. To analyze changes in chromatin structure in the promoter regions of Xist and Tsix derived from somatic cells, histone tail modifications were studied by chromatin immunoprecipitation analysis. Histones H3 and H4, which were hypoacetylated in the somatic cells, were hyperacetylated in the hybrid cells, and histone H3 lysine 4, which was hypomethylated in the somatic cells, was hypermethylated in the hybrid cells, indicating that the reactivation of Xist and Tsix was linked with chromatin modifications. In the telomeric region of DXPas34, acetylation of histones H3 and H4 was dependent on reactivation of Xist and Tsix, whereas histone H3 lysine 4 was constitutively methylated independent of the transcriptional activity of those genes. We propose that the chromatin reprogramming is linked with the resetting of the memory found in the process of choosing an active X chromosome.

Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation

“…Intriguingly, as previously reported by others (Kimura et al 2002), the promoter region of Tsix is enriched for H3-K4 dimethylation in MEFs despite the fact that Tsix is not expressed in such cells (Fig. 1B).…”

“…In these cases, the paternal and maternal alleles are distinguished by selective enrichment of the promoter region by a set of epigenetic marks associated with either active (e.g., methylation of H3 Lys 4 [H3-K4]) or silent (e.g., H3-K9 and K27 methylation) chromatin. Although some data exist concerning the chromatin features of the Xist locus (Kimura et al 2002;, a more systematic approach is clearly needed to assess the role of histone modifications in the regulation of Xist expression.…”

Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells

“…Reprogramming of the somatic nucleus to an undifferentiated state was demonstrated by reactivation of the pluripotential cell-specific marker gene Oct4-GFP and reactivation of the somatic cell-derived inactive X-chromosome. This correlated with reexpression of the undifferentiated statespecific Xist and Tsix genes (19,48). The pluripotential competence of hybrid cells was shown through the formation of teratomas following subcutaneous injection into immunodeficient mice, through contribution of the hybrid cells to normal embryogenesis in chimeras and through the reprogrammed somatic genome-derived transcription of various tissue-specific mRNAs, both in teratomas redifferentiated in vivo and in mesencephalic dopaminergic neurons redifferentiated in vitro (46,48).…”

"Nuclear Reprogramming" and "Epigenetic Reprogramming"

“…The successful establishment of human ES cells from blastocysts has also been reported [35,42,43]. An important finding is that ES cells have an intrinsic capacity for the epigenetic reprogramming of somatic genomes after cell fusion [44][45][46] (Fig. 2).…”