Early butyrate induced acetylation of histone H4 is proteoform specific and linked to methylation state

Histone posttranslational modifications (PTMs) help regulate DNA templated processes; however, relatively little work has unbiasedly explored the single-molecule combinations of histone PTMs, their dynamics on short timescales, or how these preexisting histone PTMs modulate further histone modifying enzyme activity. We use quantitative top down proteomics to unbiasedly measure histone H4 proteoforms (single-molecule combinations of PTMs) upon butyrate treatment. Our results show that histone proteoforms change in cells within 10 minutes of application of sodium butyrate. Cells recover from treatment within 30 minutes after removal of butyrate. Surprisingly, K20me2 containing proteoforms are the near-exclusive substrate of histone acetyltransferases upon butyrate treatment. Single-molecule hierarchies of progressive PTMs mostly dictate the addition and removal of histone PTMs (K16ac > K12ac ≥ K8ac > K5ac, and the reverse on recovery). This reveals the underlying single-molecule mechanism that explains the previously reported but indistinct and unexplained patterns of H4 acetylation. Thus, preexisting histone PTMs strongly modulate histone modifying enzyme activity and this suggests that proteoform constrained reaction pathways are crucial mechanisms that enable the long-term stability of the cellular epigenetic state.     

Identification of histone acetylation in a murine model of allergic asthma by proteomic analysis

The pathogenesis of asthma is closely related to histone acetylation modification, but the specific acetylation sites related to this process remain indistinct. Herein, our study sought to identify differentially modified acetylation sites and their expression distribution in cells involved in asthma in lung tissues. The airway hyper-responsiveness, inflammation, and remodeling were assessed by non-invasive whole-body plethysmography, ELISA, and hematoxylin-eosin staining to confirm the successful establishment of the allergic asthma model. Afterward, the differentially modified acetylation sites in asthmatic lung tissues were identified and validated by using proteomics and western blotting, respectively. The immunohistochemistry analysis was applied to reveal the distribution of identified acetylation sites in asthmatic lung tissues. A total of 15 differentially modified acetylation sites, including 13 upregulated (H3K9ac, H3K14ac, H3K18ac, H3K23ac,H3K27ac, H3K36ac, H2B1KK120ac, H2B2BK20ac, H2BK16ac, H2BK20ac, H2BK108ac, H2BK116ac, and H2BK120ac) and 2 downregulated (H2BK5ac and H2BK11ac) sites were identified and validated. Furthermore, immunohistochemical staining of lung tissues showed that nine of the identified histone acetylation sites (H2BK5, H2BK11, H3K18, H2BK116, H2BK20, H2BK120, H3K9, H3K36, and H3K27) were differentially expressed in airway epithelial cells, and the acetylation of identified H3 histones were observed in both eosinophil and perivascular inflammatory cells. Additionally, differential expression of histone acetylation sites was also observed in nucleus of airway epithelial cells, vascular smooth muscle cells, perivascular inflammatory cells, and airway smooth muscle cells. In conclusion, we identified potential acetylation sites associated with asthma pathogenesis. These findings may contribute greatly in the search for therapeutic approaches for allergic asthma.     

A poised chromatin platform for TGF-β access to master regulators

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-β signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.     

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z'' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.     

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.