XIST RNA Associates with Specific Regions of the Inactive X Chromatin

Abstract

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.X-chromosome inactivation in mammals is controlled by the cis-acting XIST locus, which in humans encodes a 17-kilobase untranslated RNA that is expressed only from the inactive X (Xi) 1 (1). Mouse X chromosomes with targeted deletions of the Xist gene lose the ability to undergo X inactivation (2, 3). In addition, ectopic expression of Xist RNA from multicopy transgenes during embryonic stem cell differentiation causes autosomes to exhibit features typically associated with X inactivation, such as late replication, histone H4 hypoacetylation, and transcriptional down-regulation (4, 5). These studies indicate that the Xist locus is required for X inactivation, but they do not distinguish between several possible modes of Xist action; one model is that the Xist genomic locus serves as a chromatin organizing region that, when transcribed, initiates changes in chromatin structure that are propagated in cis along the length of the Xi chromosome (6). An alternative model is that the noncoding RNA expressed from the Xist gene is functional and directly participates in the silencing process (1). One observation suggesting that the XIST transcript may directly participate in silencing comes from fluorescence in situ hybridization experiments that show that XIST RNA colocalizes with the inactive X chromosome (1, 7). However, the limited resolution of microscopy analysis does not provide any information on the molecular basis or the consequences of this colocalization.To examine the nature of the colocalization between XIST RNA and the Xi chromatin, the chromatin IP assay was used to precipitate chromatin from human female cells and then RT-PCR was performed to determine whether XIST RNA was co-precipitated. The results indicate that XIST RNA is indeed co-precipitated with chromatin by antibodies directed against several histone species. Significantly, XIST RNA was co-precipitated with antibodies against unacetylated, but not acetylated, histones H3 and H4. Given the previous finding that histone H4 is hypoacetylated at promoters of X-inactivated genes (8), these results raise the possibility that XIST RNA may be associated with promoters of silenced gen...

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“…It has been found that inactive X chromosomes in mouse cells have elevated levels of a specific histone isoform, macroH2A1.2, suggesting that this histone variant might be an effector of the silencing process [20]. This idea is supported by the observation that Xist RNA and macroH2A1.2 can form a stable ribonucleoprotein complex [21] and that the localization of macroH2A1.2 in X-chromosome chromatin depends on the expression of Xist [22]. The function of Xist RNA may be to recruit macroH2A1.2 so as to establish and maintain an inactive state [18].…”

Human BAHD1 promotes heterochromatic gene silencing

“…Indeed, HEK293 are female cells with an atypical karyotype, usually containing 3 X chromosomes, 2 of which are inactive and visible as large heterochromatic bodies (18). Through FISH, using as a probe the X-inactive specific transcript (XIST), an untranslated RNA that coats the Xi (18,19) we confirmed that exogenous BAHD1 was recruited at these 2 Xi . V5-or YFP-BAHD1 localized at the 2 Xi in HEK293 cells (Figs.…”

Nucleolin Protein Interacts with Microprocessor Complex to Affect Biogenesis of MicroRNAs 15a and 16

“…RNA Immunoprecipitation-RNA immunoprecipitation was carried out as described previously with slight modifications (30). HEK293 cells were trypsinized, washed twice with PBS, and resuspended in 10 ml of PBS.…”