Chromatin reprogramming of male somatic cell-derived XIST and TSIX 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.