Cooperation between EZH2, NSPc1-mediated histone H2A ubiquitination and Dnmt1 in HOX gene silencing

An intricate interplay between DNA methylation and polycomb-mediated gene silencing has been highlighted recently. Here we provided evidence that Nervous System Polycomb 1 (NSPc1), a BMI1 homologous polycomb protein, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in HOX gene silencing. We showed that NSPc1 stimulates H2A ubiquitination in vivo and in vitro through direct interaction with both RING2 and H2A. RT-PCR analysis revealed that loss of NSPc1, EZH2 or DNA methyltransferase 1 (Dnmt1), or inhibition of DNA methylation in HeLa cells de-represses the expression of HOXA7. Chromatin immunoprecipitation (ChIP) assays demonstrated that NSPc1, EZH2 and Dnmt1 bind to the promoter of HOXA7, which is frequently hypermethylated in tumors. Knockdown of NSPc1 results in significant reduction of H2A ubiquitination and DNA demethylation as well as Dnmt1 dissociation in the HOXA7 promoter. Meanwhile Dnmt1 deficiency affects NSPc1 recruitment and H2A ubiquitination, whereas on both cases EZH2-mediated H3K27 trimethylation remains unaffected. When EZH2 was depleted, however, NSPc1 and Dnmt1 enrichment was abolished concomitant with local reduction of H3K27 trimethylation, H2A ubiquitination and DNA methylation. Taken together, our findings indicated that NSPc1-mediated H2A ubiquitination and DNA methylation, both being directed by EZH2, are interdependent in long-term target gene silencing within cancer cells.     

H2AZ is enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment

Elucidating how chromatin influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential but unknown role during mammalian development. Here, genome-wide analysis reveals that H2AZ occupies the promoters of developmentally important genes in a manner that is remarkably similar to that of the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells, suggesting that its dynamic redistribution is necessary for cell fate transitions. Thus, H2AZ, together with PcG proteins, may establish specialized chromatin states in ES cells necessary for the proper execution of developmental gene expression programs.     

Ni(II) affects ubiquitination of core histones H2B and H2A

The molecular mechanisms of nickel-induced malignant cell transformation include effects altering the structure and covalent modifications of core histones. Previously, we found that exposure of cells to Ni(II) resulted in truncation of histones H2A and H2B and thus elimination of some modification sites. Here, we investigated the effect of Ni(II) on one such modification, ubiquitination, of histones H2B and H2A in nuclei of cultured 1HAEo- and HPL1D human lung cells. After 1-5 days of exposure, Ni(II) up to 0.25 mM stimulated mono-ubiquitination of both histones, while at higher concentrations a suppression was found. Di-ubiquitination of H2A was not affected except for a drop after 5 days at 0.5 mM Ni(II). The decrease in mono-ubiquitination coincided with the appearance of truncated H2B that lacks the K120 ubiquitination site. However, prevention of truncation did not avert the decrease of H2B ubiquitination, indicating mechanistic independence of these effects. The changes in H2B ubiquitination did not fully coincide with concurrent changes in the nuclear levels of the ubiquitin-conjugating enzymes Rad6 and UbcH6. Overall, our results suggest that dysregulation of H2B ubiquitination is a part of Ni(II) adverse effects on gene expression and DNA repair which may assist in cell transformation.     

Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells

Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.     

Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation

Histone modifications are thought to serve as epigenetic markers that mediate dynamic changes in chromatin structure and regulation of gene expression. As a model system for understanding epigenetic silencing, X chromosome inactivation has been previously linked to a number of histone modifications including methylation and hypoacetylation. In this study, we provide evidence that supports H2A ubiquitination as a novel epigenetic marker for the inactive X chromosome (Xi) and links H2A ubiquitination to initiation of X inactivation. We found that the H2A-K119 ubiquitin E3 ligase Ring1b, a Polycomb group protein, is enriched on Xi in female trophoblast stem (TS) cells as well as differentiating embryonic stem (ES) cells. Consistent with Ring1b mediating H2A ubiquitination, ubiquitinated H2A (ubH2A) is also enriched on the Xi of both TS and ES cells. We demonstrate that the enrichment of Ring1b and ubH2A on Xi is transient during TS and ES cell differentiation, suggesting that the Ring1b and ubH2A are involved in the initiation of both imprinted and random X inactivation. Furthermore, we showed that the association of Ring1b and ubH2A with Xi is mitotically stable in non-differentiated TS cells.     

RNA synthesis in the presence of transfer RNa by DNA-dependent RNA polymerase II from mouse ascites sarcoma cells

RNA polymerase II from mouse sarcoma cells catalyzed the incorporation of UMP into an acid-insoluble fraction in the presence of tRNA. This reaction was not affected by DNase or actinomycin D but was inhibited by α-amanitin. This reaction was dependent on nucleoside triphosphate and manganese ions. RNA synthesized in the presence of tRNA could be digested with RNase A. These results suggest that the RNA synthesis by RNA polymerase II from mouse sarcoma is dependent on the presence of tRNA.