The XIST noncoding RNA functions independently of BRCA1 in X inactivation

Females with germline mutations in BRCA1 are predisposed to develop breast and ovarian cancers. A previous report indicated that BRCA1 colocalizes with and is necessary for the correct localization of XIST, a noncoding RNA that coats the inactive X chromosome (Xi) to mediate formation of facultative heterochromatin. A model emerged from this study suggesting that loss of BRCA1 in female cells could reactivate genes on the Xi through loss of the XIST RNA. However, our independent studies of BRCA1 and XIST RNA revealed little evidence to support this model. We report that BRCA1 is not enriched on XIST RNA-coated chromatin of the Xi. Neither mutation nor depletion of BRCA1 causes significant changes in XIST RNA localization or X-linked gene expression. Together, these results do not support a role for BRCA1 in promoting XIST RNA localization to the Xi or regulating XIST-dependent functions in maintaining the stability of facultative heterochromatin.     

Effect of TSIX disruption on XIST expression in male ES cells

XIST and its antisense partner, TSIX, encode non-coding RNAs and play key roles in X chromosome inactivation. Targeted disruption of TSIX causes ectopic expression of XIST in the extraembryonic tissues upon maternal transmission, which subsequently results in embryonic lethality due to inactivation of both X chromosomes in females and a single X chromosome in males. TSIX, therefore, plays a crucial role in maintaining the silenced state of XIST in CIS and regulates the imprinted X inactivation in the extraembryonic tissues. In this study, we examined the effect of TSIX disruption on XIST expression in the embryonic lineage using embryonic stem (ES) cells as a model system. Upon differentiation, XIST is ectopically activated in a subset of the nuclei of male ES cells harboring the TSIX-deficient X chromosome. Such ectopic expression, however, eventually ceased during prolonged culture. It is likely that surveillance by the X chromosome counting mechanism somehow shuts off the ectopic expression of XIST before inactivation of the X chromosome.     

XIST RNA associates with specific regions of the inactive X chromatin

Microscopy studies have shown that XIST RNA colocalizes with the inactive X chromosome (Xi). However, the molecular basis for this colocalization is unknown. Here we provide two lines of evidence from chromatin immunoprecipitation experiments that XIST RNA physically associates with the Xi chromatin. First, XIST RNA can be co-precipitated by antiserum against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. The specificity of XIST RNA association with hypoacetylated chromatin, together with the previous finding that hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, raises the possibility that XIST RNA may contribute to the hypoacetylation of specific regions of the Xi so as to alter the expression of X-linked genes.     

甲基化特异性PCR检测FMR1 和XIST基因甲基化实验方法的建立

建立一种快速、灵敏的检测脆性X智障基因（Fragile X mental retardation, FMR1）和X染色体失活基因（X chromosome inactivation,XIST）甲基化的方法,用亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。以修饰后的DNA为模板,用两套不同的引物对：1对甲基化特异性引物和1对非甲基化特异性引物扩增FMR1基因（CGG）n重复序列区、FMR1 和XIST 基因的启动子区。PCR产物进一步克隆、测序。以亚硫酸氢钠和对苯二酚脱氨基修饰后的DNA为模板,进行PCR扩增后的产物与预期基因目的基因片段大小相符合,无非特异性扩增产物。测序结果表明,FMR1、XIST基因中的非甲基化的C碱基转变为U碱基,而CpG岛被甲基化的C碱基不改变。成功地建立了检测FMR1、XIST甲基化的方法,为实验室诊断脆性X综合征提供了新的方法。