Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation,but It Alters Nucleosome Stability and Histone H1 Binding

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl₂ concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.     

Histone H2A Ubiquitination Inhibits the Enzymatic Activity of H3 Lysine 36 Methyltransferases

Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.     

A conserved function for the H2A.Z C terminus

Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.     

Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46

Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.     

DNA methylation and demethylation in mammals

Cell type-specific DNA methylation patterns are established during mammalian development and maintained in adult somatic cells. Understanding how these patterns of 5-methylcytosine are established and maintained requires the elucidation of mechanisms for both DNA methylation and demethylation. The enzymes involved in the de novo methylation of DNA and the maintenance of the resulting methylation patterns have been fairly well characterized. However, important remaining challenges are to understand how DNA methylation systems function in vivo and in the context of chromatin. In addition, the enzymes and mechanisms for demethylation remain to be elucidated. There is still no consensus as to how active enzymatic demethylation is achieved in mammalian cells, but recent studies implicate base excision repair for genome-wide DNA demethylation in germ cells and early embryos.     

Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.