Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro

Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3.     

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.     

Identification of a novel histone H3 specific protease activity in nuclei of chicken liver

Evolutionary conserved histone proteins play a very important role in the regulation of eukaryotic gene expression by undergoing post translational modifications within the tail regions. However, their role in tissue-specific gene expression and development remains unclear. In this study, we provide evidence for in vivo tissue-specific proteolytic cleavage of histone H3 in the liver of adult white Leghorn chickens, which we believe to be regulated by tissue-specific protease activity and epigenetic markers. The cleavage of histone H3 in the liver of adult chickens is very unique, and can serve as a model for studying tissue-specific changes in chromatin organization and gene expression. For the first time, we have identified and partially purified histone H3-specific protease activity that is distinct from histone H3 protease activities recently reported. Together, our data provide evidence of proteolytic processing and identification of protease activity that is specific to histone H3 in the liver of adult chickens, which may be involved in the regulation of gene expression during development, aging, and age-associated diseases.     

Histone monoubiquitylation position determines specificity and direction of enzymatic cross-talk with histone methyltransferases Dot1L and PRC2

It is well established that chromatin is a destination for signal transduction, affecting many DNA-templated processes. Histone proteins in particular are extensively post-translationally modified. We are interested in how the complex repertoire of histone modifications is coordinately regulated to generate meaningful combinations of "marks" at physiologically relevant genomic locations. One important mechanism is "cross-talk" between pre-existing histone post-translational modifications and enzymes that subsequently add or remove modifications on chromatin. Here, we use chemically defined "designer" nucleosomes to investigate novel enzymatic cross-talk relationships between the most abundant histone ubiquitylation sites, H2AK119ub and H2BK120ub, and two important histone methyltransferases, Dot1L and PRC2. Although the presence of H2Bub in nucleosomes greatly stimulated Dot1L methylation of H3K79, we found that H2Aub did not influence Dot1L activity. In contrast, we show that H2Aub inhibited PRC2 methylation of H3K27, but H2Bub did not influence PRC2 activity. Taken together, these results highlight how the position of nucleosome monoubiquitylation affects the specificity and direction of cross-talk with enzymatic activities on chromatin.     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

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.     

p300-mediated Acetylation of Histone H3 Lysine 56 Functions in DNA Damage Response in Mammals

The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis.     

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.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.     

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.     

Purification and characterization of a novel histone H2A specific protease (H2Asp) from chicken liver nuclear extract

The proteolysis of the N- or the C-terminal tails of histones have recently emerged as a novel form of irreversible posttranslational modifications of histones. However, there are very few reports describing purification of a histone specific protease. Here, we report a histone H2A specific protease (H2Asp) activity in the chicken liver nuclear extract. The H2Asp was purified to homogeneity and was found to be a ~ 10.5 kDa protein. It demonstrated high specificity to histone H2A and was an aspartic acid like protease as shown by protease inhibition assay. The H2Asp, in the in vitro cleavage assay generated a single clipped H2A product which comigrated along with histone H4 in the SDS-PAGE and migrated as a single band when single H2A was used as substrates. The expression of H2Asp was independent of age and was tissue specific, which was demonstrated only in the nuclear extracts of chicken liver and not from the same of other tissues like brain, muscles and erythrocytes. It was also seen that H2Asp activity also exists in other classes of vertebrates from Pisces to Mammals. This report forms the first such report describing purification of a histone H2A specific protease.     

Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH₂-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH₂-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH₂-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.