Transcriptional repression of Xi Xist Transcription of Xist Xist RNA Spreading of Xist Recruitment of repression factors. Stable repression Translocated Xic cannot efficiently silence autosome regions. A XX ES cell line with T(X;4)37H translocation 12
The spread of mh3k27 into chromosome 4 region is inefficient. H3K27me3 is largely confined to the X chromosome. TMA: differentiated ES cells T37H: adult fibroblasts Histone acetylation (ach4) mark is maintained on chromosome 4. 13
Xist RNA is largely limited to X chromosome. Mapping of the translocation breakpoint X-chromosome is enriched for LINE (long interspersed element) Translocation breakpoint The block to Xist spreading coincides with a region high in gene content but low in LINE. 14
Cytosoine methylation contributes to the maintenance of X inactivation. CCGCGG CCGG c/c: Dnmt1 -/- Emb: embryo YS: yolk sac Exon 1 of Xist Xa Xi Xa Y Xist Gene subject to X-inactivation Cytosine methylation In the absence of cytosine methylation, X inactivation in the embryo is initiated, but not stably maintained. WT E8.5 lacz Dnmt c/c E8.5 X-inactivation is initiated normally in embryo. lacz Dnmt c/c E9.5 X-inactivation is not maintained. lacz lacz LacZ is on the paternal allele. 15
Imprinted X inactivation in extraembryonic tissue does not require cytosine methylation. Imprinted X-inactivation in extraembryonic tissue Msg1 promoter lacz Maternal Paternal ve: visceral endoderm, part of extraembryonic tissue SmcHD1 is critical for X-inactivation. Structural maintenance of chromosome hinge domain Identified as a modifier of transgene silencing Homozygous mutation of SmcHD1 leads to lethality in females. MommeD1: ENU-induced mouse mutant SmcHD1 gene trap (gt): inactivation of SmcHD1 by marker integration 16
X-inactivation is impaired in SmcHD1 -/- mutant. E7.5 Embryo Placenta SmcHD1 + Xist + GFP + Xist - SmcHD1 - Xist + GFP + Xist - P M P M E10.5 Xist activation and H3K27me3 localization appear normal in SmcHD1 Homozygous mutant. 17
SmcHD1 localizes to the inactive X chromosome. Reactivation of genes on Xi is accompanied by DNA demethylation. 18
H3K27me3 is dispensable for random X inactivation, but is required for the maintenance of imprinted X inactivation. Xist Tsix GFP Tsix mutation leads to strong expression of Xist and preferential X-inactivation of GFP marked chromosome. Eed is part of the complex that catalyzes H3K27me3. H3K27me3 is undetectable in Eed -/- embryos. At E5.5, extraembryonic tissues have undergone imprinted X-inactivation (paternal X with GFP), while both X chromosomes remain active in embryo. The imprinted X-inactivation is erased in the embryonic epiblast cells at this stage. Imprinted X-inactivation is initiated normally in the absence of Eed, but X-inactivation is not stably maintained in some trophoblast cells (TB). Random X inactivation is normally initiated and maintained in the embryo in the absence of Eed; but imprinted X-inactivation is not maintained. 19
Monoallelic expression of X-linked genes in Eed embryos. Some genes (15%) escape X-inactivation in human. Pseudo autosome genes Significant expression from Xi Silenced on Xi Expression measured from 9 lines of human/rodent hybrid cells containing Xi. Each line represents data from one hybrid cell line. 20
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