Extraction of histones H2A,H3 and H4 from yeast nuclei: Measurement of the extent of yeast histone acetylation following one-dimensional gel electrophoresis

Yeast histones H2A, H3 and H4 were specifically extracted from purified nuclei using a 2% NaCl/75% ethanol solution. The extraction resulted in the complete removal of H2A, H3 and H4 from the nuclear pellet, as monitored by SDS-polyacrylamide gel electrophoresis of the protein. The relative absence of nonhistone proteins from this histone subset simplifies the determination of the extent of histone modification in yeast. Levels of H4 acetylation were measured directly on Coomassie blue-stained Triton acid-urea gels and the levels verified by gel fluorography of the [³H]acetate-labeled histone.     

Mammalian target of rapamycin complex 2 (mTORC2) controls glycolytic gene expression by regulating Histone H3 Lysine 56 acetylation

Metabolic reprogramming is a hallmark of cancer cells, but the mechanisms are not well understood. The mammalian target of rapamycin complex 2 (mTORC2) controls cell growth and proliferation and plays a critical role in metabolic reprogramming in glioma. mTORC2 regulates cellular processes such as cell survival, metabolism, and proliferation by phosphorylation of AGC kinases. Components of mTORC2 are shown to localize to the nucleus, but whether mTORC2 modulates epigenetic modifications to regulate gene expression is not known. Here, we identified histone H3 lysine 56 acetylation (H3K56Ac) is regulated by mTORC2 and show that global H3K56Ac levels were downregulated on mTORC2 knockdown but not on mTORC1 knockdown. mTORC2 promotes H3K56Ac in a tuberous sclerosis complex 1/2 (TSC1/2) mediated signaling pathway. We show that knockdown of sirtuin6 (SIRT6) prevented H3K56 deacetylation in mTORC2 depleted cells. Using glioma model consisting of U87EGFRvIII cells, we established that mTORC2 promotes H3K56Ac in glioma. Finally, we show that mTORC2 regulates the expression of glycolytic genes by regulating H3K56Ac levels at the promoters of these genes in glioma cells and depletion of mTOR leads to increased recruitment of SIRT6 to these promoters. Collectively, these results identify mTORC2 signaling pathway positively promotes H3K56Ac through which it may mediate metabolic reprogramming in glioma.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

Induction by Fructose Force-Feeding of Histone H3 and H4 Acetylation at Their Lysine Residues around the Slc2a5 Gene and Its Expression in Mice

It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.     

Protein-arginine methyltransferase 1 (PRMT1) methylates Ash2L, a shared component of mammalian histone H3K4 methyltransferase complexes

Multiple enzymes and enzymatic complexes coordinately regulate the addition and removal of post-translational modifications on histone proteins. The oncoprotein Ash2L is a component of the mixed lineage leukemia (MLL) family members 1-4, Setd1A, and Setd1B mammalian histone H3K4 methyltransferase complexes and is essential to maintain global trimethylation of histone H3K4. However, regulation of these complexes at the level of expression and activity remains poorly understood. In this report, we demonstrate that Ash2L is methylated on arginine residues both in vitro and in cells. We found that both protein-arginine methyltransferases 1 and 5 methylate Arg-296 within Ash2L. These findings are the first to demonstrate that post-translational modifications occur on the Ash2L protein and provide a novel example of cross-talk between chromatin-modifying enzyme complexes.     

Synthesis and chemiluminescence of copolymers of 5-amino-8-vinyl-phthalazine-1, 4(2H,3H)-dione with methyl methacrylate or styrene,and of α, ω-bis[5-amino-phthalazine-1,4 (2H,3H)-dion-]8-yl alkanes [=α, ω-bis(6-luminyl) alkanes]: Investigations on an intramolecular ‘distance effect’

Oligomers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione exhibit about 0.05% of the chemiluminescence quantum yield of the corresponding ‘monomer unit’, i.e. 5-amino-8-ethyl-phthalazine-1,4(2H,3H)-dione which has a similar quantum yield to luminol. The quantum yields of copolymers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione (1a) with methyl methacrylate or with styrene increase up to 1000-fold, relative to the quantum yield of oligomers of (1a). Thus the monomer units of methyl methacrylate or styrene appear to act as ‘spacers’ between the lumigenic groups. α,ω-Bis[(5-amino-phthalazine-1,4(2H,3H)-dion-)8-yl] alkanes show an analogue ‘distance’ effect: the chemiluminescence quantum yield increases with increasing alkane chain length. As the fluorescence of the corresponding amino phthalates (which are intermediates in the synthesis of the phthalazine diones) is only slightly influenced by the distance between the lumigenic groups it is suggested that a mainly chemical ‘distance effect’ is working here: the smaller the intramolecular distance between the hydrazide groups the more inhibition exists in respect of the oxidative reaction producing the luminol-type chemiluminescence.