Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies (original) (raw)
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Identification of histone H3 lysine 36 acetylation as a highly conserved histone modification
The Journal of biological chemistry, 2007
Histone lysine (K) acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an "acetyl/methyl switch" governing chromatin function along transcription units.
A direct link between core histone acetylation and transcriptionally active chromatin
The EMBO Journal, 1988
An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope e-N-acetyl lysine. Affinity-purified antibodies were used to fractionate oligoand mononucleosomal chromatin fragments from the nuclei of 15-day chicken embryo erythrocytes. Antibodybound chromatin was found to contain elevated levels of acetylated core histones. On probing with sequences of a D globin, an actively transcribed gene, the antibodybound chromatin was 15to 30-fold enriched relative to the input chromatin. Using ovalbumin sequences as a probe, no enrichment was observed. The results demonstrate directly that transcriptionally active genes carry acetylated core histones.
Quality of histone modification antibodies undermines chromatin biology research
Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains would help to improve the reproducibility of experimental work in this field.
Influence of combinatorial histone modifications on antibody and effector protein recognition
Current biology : CB, 2011
Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.
Patterns of histone acetylation
European Journal of Biochemistry, 1990
The N-terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. To increase understanding of the significance of this modification we have studied the specificity of site utilisation in the monoacetyl, diacetyl and triacetyl forms of histones H3, H4 and H2B (histone H2A has only a single modification site), using pig thymus and HeLa cells as the source of histones. The HeLa histones were extracted from cells grown both with and without butyrate treatment. It is found that for histone H3 there is a fairly strict order of site occupancy: Lysl4, followed by Lys23, followed by Lysl8 in both pig and HeLa histones. Since the order and specificity is the same when butyrate is added to the HeLa cell cultures, we conclude that addition of the fatty acid does not scramble the specificity of site utilisation, but merely allows more of the natural forms of modified histone to accumulate. For histone H4, the monoacetyl form is exclusively modified at Lysl6, but further addition of acetyl groups is less specific and progresses through sites 12, 8 and 5 in an N-terminal direction. Similar results were obtained for H4 from both pig thymus and butyratetreated HeLa cells. Histone H2B could be studied in detail only from butyrate-treated HeLa cells and a much lower level of site specificity was found: sites 12 and 15 were preferred to the more Nand C-terminal sites at Lys5 and Lys20. The data reinforces the view that lysine acetylation in core histones is a very specific phenomenon that plays several functionally distinct roles. The high degree of site specificity makes it unlikely that the structural effects of acetylation are mediated merely by a generalised reduction of charge in the histone N-terminal domains.
bioRxiv (Cold Spring Harbor Laboratory), 2022
The ATPase family, AAA domain containing 2B (ATAD2B) protein has a C-terminal bromodomain that functions as a 'reader' of acetylated lysine residues on histone proteins. However, the molecular mechanisms by which it recognizes chromatin with multiple post-translational modifications remain poorly understood. To gain insights into the recognition of acetylated lysines by the ATAD2B bromodomain, we investigated the recognition of combinatorial histone H4 post-translational modifications using structural and functional approaches. Using isothermal titration calorimetry, we assessed the binding affinities of the ATAD2B bromodomain with distinct histone H4 peptides harboring multiple modifications. Our results show that the ATAD2B bromodomain selectively. CC-BY-NC-ND 4.
Analytical and bioanalytical chemistry, 2016
We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R (2) ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the...
Acetylation of Histone H3 at the Nucleosome Dyad Alters DNA-Histone Binding
Journal of Biological Chemistry, 2009
Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys 3 Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly. . 3 The abbreviations used are: PTM, post-translational modification; BZA, benzamidine; EPL, expressed protein ligation; H3-(1-109), residues 1-109 of histone H3, generated as a thioester; H3Pep, peptide with sequence CAIHAKRVTIMPKDIQLARRIRGERA; H3, full-length histone H3 protein; HO, histone octamer; mp2, modified high affinity 601 positioning sequence; MESNA, mercaptoethanesulfonic acid; RP-HPLC, reverse phase HPLC; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flightmass spectrometry; ExoIII, exonuclease III.