Differential loss of histone H3 isoforms mono-, di- and tri-methylated at lysine 4 during X-inactivation in female embryonic stem cells.

Silencing of genes on one of the two female X chromosomes early in development helps balance expression of X-linked genes between XX females and XY males and involves chromosome-wide changes in histone variants and modifications. Mouse female embryonic stem (ES) cells have two active Xs, one of which is silenced on differentiation, and provide a powerful model for studying the dynamics of X inactivation. Here, we use immunofluorescence microscopy of metaphase chromosomes to study changes in H3 mono-, di- or tri-methylated at lysine 4 (H3K4mel, -2 or -3) on the inactivating X (Xi) in female ES cells. H3K4me3 is absent from Xi in approximately 25% of chromosome spreads by day 2 of differentiation and in 40-50% of spreads by days 4-6, making it one of the earliest detectable changes on Xi. In contrast, loss of H3K4me2 occurs 1-2 days later, when histone acetylation also diminishes. Remarkably, H3K4mel is depleted on both (active) X chromosomes in undifferentiated female ES cells, and on the single X in males, and remains depleted on Xi. Consistent with this, chromatin immunoprecipitation reveals differentiation-related reductions in H3K4me2 and H3K4me3 at the promoter regions of genes undergoing X-inactivation in female ES cells, but no comparable change in H3K4me1.

Human CD34+ hematopoietic progenitor cells hyperacetylate core histones in response to sodium butyrate, but not trichostatin A.

Cells positive for the cell surface marker CD34 from bone marrow or umbilical cord blood form a subset of quiescent, hematopoetic precursors that can establish human hematopoesis in immunodeficient mice and can progress down various differentiation pathways in vitro. They provide a valuable model system in which progression from quiescent to cycling to differentiated states can be linked to changes in chromatin and histone modification. We have used the deacetylase inhibitor sodium butyrate to show that turnover of histone H4 acetates is rapid and comparable in quiescent and cycling CD34+ cells from human umbilical cord blood (CD34+ UBC). Surprisingly, the widely used inhibitor trichostatin A (TSA) had little (cycling cells) or no (quiescent cells) effect on H4 acetylation in CD34+ UBC. Among five cell types examined, CD34+ UBC were unique in expressing all (putative) deacetylases tested (HDAC1, -2, -3, -4, -6, -7, and -8 and SIRT1-4), but no single deacetylase correlated with their TSA resistance. Also, HDAC1, -2, -3, and -6 complexes isolated from CD34+ UBC by immunoprecipitation were all inhibited by TSA in vitro. Thus, TSA resistance of CD34+ UBC is not due to acquired or intrinsic TSA resistance of their deacetylases and may reflect an enhanced ability to process the drug.

Molecular and cytogenetic analysis of the spreading of X inactivation in X;autosome translocations.

We have performed detailed studies of the spreading of X inactivation in five unbalanced human X;autosome translocations. Using allele-specific RT-PCR we observed long-range silencing of autosomal genes located up to 45 Mb from the translocation breakpoint, directly demonstrating the ability of X inactivation to spread in cis through autosomal DNA. Spreading of gene silencing occurred in either a continuous or discontinuous fashion in different cases, suggesting that some autosomal DNA is resistant to the X inactivation signal. This spread of inactivation was accompanied by, but not dependent upon, CpG island methylation. Observations of late-replication, histone acetylation and histone methylation show that X inactivation can spread in the absence of cytogenetic features normally associated with the inactive X. However, the distribution of histone modifications which distinguish the inactive X are more accurate cytogenetic measures of the spread of X inactivation than late-replication. Overall, despite remarkable variation in the spread of X inactivation among the five cases there was good correlation between the pattern of gene silencing and the attenuation of clinical phenotype associated with each partial autosomal trisomy. We discuss our observations in the context of hypotheses which address the spread of X inactivation.

X-linked genes in female embryonic stem cells carry an epigenetic mark prior to the onset of X inactivation.

We use chromatin immunoprecipitation to show that genes on the two active X chromosomes in undifferentiated, XX female embryonic stem cells (ES cells) are marked by hyperacetylation of all core histones, hyper(di)methylation of H3 lysine 4 and hypo(di)methylation of H3 lysine 9, compared with autosomal genes or genes on the single active X in XY male cells. The mark is found on both coding and promoter regions. On differentiation, and after the onset of X inactivation, the mark is reversed on the inactive X, whose genes show extreme hypoacetylation of all four core histones, hypo(di)methylation of H3K4 and hyper(di)methylation of H3K9. The mark is retained on the active X in female ES cells for at least several days of differentiation, but is not present in adult females. The selective marking of X-linked genes in female ES cells in a way that distinguishes them from the equivalent genes in males, is unprecedented. We suggest that the mark forms part of a chromatin-based mechanism that restricts X-inactivation to cells with more than one X chromosome.