Lysine Residues Of Histone H3 Research Articles

Histone H3 N-Terminal Lysine Acetylation Governs Fungal Growth, Conidiation, and Pathogenicity through Regulating Gene Expression in Fusarium pseudograminearum.

The acetylation of histone lysine residues regulates multiple life processes, including growth, conidiation, and pathogenicity in filamentous pathogenic fungi. However, the specific function of each lysine residue at the N-terminus of histone H3 in phytopathogenic fungi remains unclear. In this study, we mutated the N-terminal lysine residues of histone H3 in Fusarium pseudograminearum, the main causal agent of Fusarium crown rot of wheat in China, which also produces deoxynivalenol (DON) toxins harmful to humans and animals. Our findings reveal that all the FpH3K9R, FpH3K14R, FpH3K18R, and FpH3K23R mutants are vital for vegetative growth and conidiation. Additionally, FpH3K14 regulates the pathogen's sensitivity to various stresses and fungicides. Despite the slowed growth of the FpH3K9R and FpH3K23R mutants, their pathogenicity towards wheat stems and heads remains unchanged. However, the FpH3K9R mutant produces more DON. Furthermore, the FpH3K14R and FpH3K18R mutants exhibit significantly reduced virulence, with the FpH3K18R mutant producing minimal DON. In the FpH3K9R, FpH3K14R, FpH3K18R, and FpH3K23R mutants, there are 1863, 1400, 1688, and 1806 downregulated genes, respectively, compared to the wild type. These downregulated genes include many that are crucial for growth, conidiation, pathogenicity, and DON production, as well as some essential genes. Gene ontology (GO) enrichment analysis indicates that genes downregulated in the FpH3K14R and FpH3K18R mutants are enriched for ribosome biogenesis, rRNA processing, and rRNA metabolic process. This suggests that the translation machinery is abnormal in the FpH3K14R and FpH3K18R mutants. Overall, our findings suggest that H3 N-terminal lysine residues are involved in regulating the expression of genes with important functions and are critical for fungal development and pathogenicity.

LSD1 inhibitors for cancer treatment: Focus on multi-target agents and compounds in clinical trials.

Histone lysine-specific demethylase 1 (LSD1/KDM1A) was first identified in 2004 as an epigenetic enzyme able to demethylate specific lysine residues of histone H3, namely H3K4me1/2 and H3K9me1/2, using FAD as the cofactor. It is ubiquitously overexpressed in many types of cancers (breast, gastric, prostate, hepatocellular, and esophageal cancer, acute myeloid leukemia, and others) leading to block of differentiation and increase of proliferation, migration and invasiveness at cellular level. LSD1 inhibitors can be grouped in covalent and non-covalent agents. Each group includes some hybrid compounds, able to inhibit LSD1 in addition to other target(s) at the same time (dual or multitargeting compounds). To date, 9 LSD1 inhibitors have entered clinical trials, for hematological and/or solid cancers. Seven of them (tranylcypromine, iadademstat (ORY-1001), bomedemstat (IMG-7289), GSK-2879552, INCB059872, JBI-802, and Phenelzine) covalently bind the FAD cofactor, and two are non-covalent LSD1 inhibitors [pulrodemstat (CC-90011) and seclidemstat (SP-2577)]. Another TCP-based LSD1/MAO-B dual inhibitor, vafidemstat (ORY-2001), is in clinical trial for Alzheimer's diseases and personality disorders. The present review summarizes the structure and functions of LSD1, its pathological implications in cancer and non-cancer diseases, and the identification of LSD1 covalent and non-covalent inhibitors with different chemical scaffolds, including those involved in clinical trials, highlighting their potential as potent and selective anticancer agents.

Set1, Set2, and the Clock‐cycle complex are necessary to prevent age‐related retinal degeneration in Drosophila melanogaster

Age‐related eye disease is a prevalent problem that is exacerbated by the rising, aging population. Drosophila melanogaster serves as a powerful model organism for studying age‐related eye disease, as they share 60% of their genetic material with humans. Here, we seek to understand how changes in gene expression regulate photoreceptor neuron survival during aging. A preliminary RNA interference screen was carried out to identify genes necessary for cell survival throughout aging. Microscopy imaging and reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) were used to further investigate the role of histone methyltransferases Set1 and Set2 and the transcription factor Clock in photoreceptor neuron survival. Set1 and Set2 add methyl groups to lysine residues of histone H3, while Clock forms a heterodimer with the transcription factor cycle to activate transcription of genes involved in the circadian rhythm. Microscopy imaging was used to qualitatively and quantitatively assess loss of rhabdomeres, compartments of photoreceptor cells responsible for phototransduction, in flies lacking expression of either Set1, Set2, or a functional Clock‐cycle complex. Flies expressing RNA interference against Set1 and Set2 show retinal degeneration at or before the age of 30 days. Flies that fail to form a functional Clock‐cycle complex due to expression of a dominant negative form of Clock (ClkDN) show progressive retinal degeneration starting at the age of 5 days. Interestingly, this retinal degeneration is rescued when flies are raised in the dark. These phenotypes were validated by measuring target gene expression using RT‐qPCR. RNA interference against mCherry and overexpression of LacZ were used as controls for Set1 and Set2 RNA interference and overexpression of ClkDN, respectively. These findings suggest that histone methylation and Clock‐dependent gene expression play a role in age‐related eye disease in both Drosophila and humans.

SMYD5 is a histone H3-specific methyltransferase mediating mono-methylation of histone H3 lysine 36 and 37

Although post-translational modifications (-PTMs) of some histone H3 lysine residues are well studied, the PTMs of histone H3 lysine 37 in mammalian cells remain largely unknown. In this study, we provide evidence to show that SMYD family member 5 (SMYD5) is a histone H3-specfic methyltransferase that catalyzes mono-methylation of H3 lysine 36 and 37 (H3K36/K37me1) in vitro. The site-mutagenesis analysis shows that a species-conserved histidine in its catalytic SET domain is required for its histone methyltransferase activity. Genetic deletion of Smyd5 in murine embryonic stem cells (mESCs) partially reduces the global histone H3K37me1 level in cells, suggesting SMYD5 is one of histone methyltransferases catalyzing histone H3K37me1 in vivo. Hence, our study reveals that SMYD5 is a histone H3-specific methyltransferase that mediates histone H3K36/K37me1, which provides a biochemical basis for further studying its functions in mammalian cells.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

HD2-type histone deacetylases: unique regulators of plant development and stress responses.

Plants have developed sophisticated and complex epigenetic regulation-based mechanisms to maintain stable growth and development under diverse environmental conditions. Histone deacetylases (HDACs) are important epigenetic regulators in eukaryotes that are involved in the deacetylation of lysine residues of histone H3 and H4 proteins. Plants have developed a unique HDAC family, HD2, in addition to the RPD3 and Sir2 families, which are also present in other eukaryotes. HD2s are well conserved plant-specific HDACs, which were first identified as nucleolar phosphoproteins in maize. The HD2 family plays important roles not only in fundamental developmental processes, including seed germination, root and leaf development, floral transition, and seed development but also in regulating plant responses to biotic and abiotic stresses. Some of the HD2 members coordinate with each other to function. The HD2 family proteins also show functional association with RPD3-type HDACs and other transcription factors as a part of repression complexes in gene regulatory networks involved in environmental stress responses. This review aims to analyse and summarise recent research progress in the HD2 family, and to describe their role in plant growth and development and in response to different environmental stresses.

Deacetylation of H4 lysine16 affects acetylation of lysine residues in histone H3 and H4 and promotes transcription of constitutive genes

ABSTRACT Histone modification map of H4 N-terminal tail residues in Saccharomyces cerevisiae reveals the prominence of lysine acetylation. Previous reports have indicated the importance of lysine acetylation in maintaining chromatin structure and function. H4K16, a residue with highly regulated acetylation dynamics has unique functions not overlapping with the other H4 N- terminal acetylable residues. The present work unravels the role of H4K16 acetylation in regulating expression of constitutive genes. H4K16 gets distinctly deacetylated over the coding region of constitutively expressed genes. Deacetylation of H4K16 reduces H3K9 acetylation at the cellular and gene level. Reduced H3K9 acetylation however did not negatively correlate with active gene transcription. Significantly, H4K16 deacetylation was found to be associated with hypoacetylated H4K12 throughout the locus of constitutive genes. H4K16 and K12 deacetylation is known to favour active transcription. Sas2, the HAT mutant showed similar patterns of hypoacetylated H3K9 and H4K12 at the active loci, clearly implying that the modifications were associated with deacetylation state of H4K16. Deacetylation of H4K16 was also concurrent with increased H3K56 acetylation in the promoter region and ORF of the constitutive genes. Combination of all these histone modifications significantly reduced H3 occupancy, increased promoter accessibility and enhanced RNAPII recruitment at the constitutively active loci. Consequently, we found that expression of active genes was higher in H4K16R mutant which mimic deacetylated state, but not in H4K16Q mimicking constitutive acetylation. To summarize, H4K16 deacetylation linked with H4K12 and H3K9 hypoacetylation along with H3K56 hyperacetylation generate a chromatin landscape that is conducive for transcription of constitutive genes.

Quantification of Proteins and Histone Marks in Drosophila Embryos Reveals Stoichiometric Relationships Impacting Chromatin Regulation.

Gene transcription in eukaryotes is regulated through dynamic interactions of a variety of different proteins with DNA in the context of chromatin. Here, we used mass spectrometry for absolute quantification of the nuclear proteome and methyl marks on selected lysine residues in histone H3 during two stages of Drosophila embryogenesis. These analyses provide comprehensive information about the absolute copy number of several thousand proteins and reveal unexpected relationships between the abundance of histone-modifying and -binding proteins and the chromatin landscape that they generate and interact with. For some histone modifications, the levels in Drosophila embryos are substantially different from those previously reported in tissue culture cells. Genome-wide profiling of H3K27 methylation during developmental progression and in animals with reduced PRC2 levels illustrates how mass spectrometry can be used for quantitatively describing and comparing chromatin states. Together, these data provide a foundation toward a quantitative understanding of gene regulation in Drosophila.

Stoichiometry of Multi‐protein Complexes containing RTT109, Vps75, and Histone H3–H4

RTT109 is a fungal‐specific acetyltransferase that acetylates lysine residues in histone H3–H4. These acetylations play a critical role in nucleosome dynamics as required for DNA transcription, replication and repair. RTT109 acetylation of specific histone H3 lysine residues requires Vps75. Vps75 is a histone chaperone belonging to the Nap‐family; it directly binds the RTT109 enzyme and the histone H3–H4 substrate. The goal of this study was to determine the absolute stoichiometry of the active complex containing RTT109, Vps75, and histone H3–H4. The stoichiometry was unclear as a Vps75 homodimer can crystalize with either one or two copies of RTT109. A high‐resolution structure of the complex between Vps75 and histone H3–H4 is not available, and both Vps75 and histone H3–H4 are known to form oligomers in solution. To measure absolute stoichiometry, we have used an elegant series of analytical ultracentrifugation experiments. We characterize all possible combinations of RTT109, Vps75, and histone H3–H4. These centrifugation experiments are complemented by other biophysical approaches. Our final stoichiometry, combined with targeted mutagenesis, reveals the organization of the active complex containing RTT109, Vps75, and histone H3–H4. Our results pave the way for high‐resolution structural studies and have repercussions for the mechanism and preferred substrate of RTT109 bound by Vps75.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chromatin-associated regulation of sorbitol synthesis in flower buds of peach.

PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.

KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation.

Kaposi’s sarcoma associated herpesvirus (KSHV) persists in a highly-ordered chromatin structure inside latently infected cells with the majority of the viral genome having repressive marks. However, upon reactivation the viral chromatin landscape changes into ‘open’ chromatin through the involvement of lysine demethylases and methyltransferases. Besides methylation of lysine residues of histone H3, arginine methylation of histone H4 plays an important role in controlling the compactness of the chromatin. Symmetric methylation of histone H4 at arginine 3 (H4R3me2s) negatively affects the methylation of histone H3 at lysine 4 (H3K4me3), an active epigenetic mark deposited on the viral chromatin during reactivation. We identified a novel binding partner to KSHV viral DNA processivity factor, ORF59-a protein arginine methyl transferase 5 (PRMT5). PRMT5 is an arginine methyltransferase that dimethylates arginine 3 (R3) of histone H4 in a symmetric manner, one hallmark of condensed chromatin. Our ChIP-seq data of symmetrically methylated H4 arginine 3 showed a significant decrease in H4R3me2s on the viral genome of reactivated cells as compared to the latent cells. Reduction in arginine methylation correlated with the binding of ORF59 on the viral chromatin and disruption of PRMT5 from its adapter protein, COPR5 (cooperator of PRMT5). Binding of PRMT5 through COPR5 is important for symmetric methylation of H4R3 and the expression of ORF59 competitively reduces the association of PRMT5 with COPR5, leading to a reduction in PRMT5 mediated arginine methylation. This ultimately resulted in a reduced level of symmetrically methylated H4R3 and increased levels of H3K4me3 marks, contributing to the formation of an open chromatin for transcription and DNA replication. Depletion of PRMT5 levels led to a decrease in symmetric methylation and increase in viral gene transcription confirming the role of PRMT5 in viral reactivation. In conclusion, ORF59 modulates histone-modifying enzymes to alter the chromatin structure during lytic reactivation.

Abstract 1376: Novel HDAC inhibitor SBAK-GHA: potential therapuetic molecule for lymophocytic leukaemia

Abstract In cancer cells, an imbalance exists between Histone acetylase(HAT) and Histone deacetylase(HDAC) activities and current research focuses actively on seeking competitive HDAC inhibitors (HDACi) for chemotherapeutic intervention.We have designed and synthesized a library of novel HDACi molecules from naturally occurring boswellic acids as an alternate CAP for already validated pharmacophore of suberanilohydroxamic acid (SAHA).Novel HDACi molecules were screened against a panel of cancer cell lines. A lead HDACi i.e.N1-hydroxy-N5-(3-α-hydroxy-11-oxo-24-norurs-12-en-4-yl) glutaramide (SBAK-GHA) having IC50 of 1.5µM against leukaemia HL-60 cell line and 6µM in enzyme based assay was obtained. Further HDACi, isoform evaluation was done for SBAK-GHA and it was found to be HDAC Class 1 Specific, especially against HDAC 2 (IC50 of 200 nM) and HDAC 8(IC50 of 250 nM). Acetylation level of different histone residues on H3 and H4 upon SBAK-GHA treatment in HL-60 cell line was studied by immunoblotting. HDACi, SAHA and Sodium Butyrate (NaB) were used as positive controls to compare acetylation levels of different H3 and H4 residues.SAHA and NaB showed significant increase in H3K9, H3K14, H3K27, H4K5, H4K12, and H4K16 acetylation levels while H3K18 showed mild change in acetylation levels. On the other hand SBAK-GHA showed significant increase in acetylation levels of all the lysine residues of histone H3 and H4 especially H4K16 in comparison to SAHA and NaB. In order to analyse varied expression of cyclins and their corresponding control of different phases of cell cycle, we performed chromatin immunoprecipitation assay (ChIP) and observed acetylation pattern of different histone (H3 and H4) residues regulates cyclins differently. In order to analyse the correlation of acetylation on different H3 and H4 residues and their varied pattern of regulation of cyclins, we next analyzed the effect of SBAK-GHA on different cell cycle events in HL-60 cells and found G1 phase cell cycle arrest at significantly low concentration (1µM).We further observed SBAK-GHA induced apoptosis at lower concentrations while as autophagy was found at higher concentrations. Furthermore,we conducted mitochondrial membrane potential studies of SBAK-GHA in HL-60 cells and observed significant loss in mitochondrial membrane potential. We took our findings from in vitro studies and analysed effect of SBAK-GHA on different in vivo murine models. SBAK-GHA showed significant activity especially against P388 lymphocytic leukemia compared to SAHA and 5-FU. Moreover, SBAK-GHA showed promising activity against Ehrlich Ascites Carcinoma (EAC). In conclusion our findings indicate that our lead molecule of interest have great therapeutic potential singly or in combination, however further mechanism of action needs to be elucidated before taking it from Bench to Bedside. Note: This abstract was not presented at the meeting. Citation Format: Javeed Ahmad Bhat, Mudassier Ahmad, Nawab John Dar, Aashiq Hussain, Rauf Ahmad Najar, Simmi Sharma, Mubashir Javeed Minto, Dilip Manikrao Mondhe, Vaqar M. Adhami, Bhahwal Ali Shah, Neena caplash, Abid Hamid*. Novel HDAC inhibitor SBAK-GHA: potential therapuetic molecule for lymophocytic leukaemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1376. doi:10.1158/1538-7445.AM2017-1376

Association between H3K4me3/BDNF and the cognitive function of workers occupationally exposed to aluminum

Objective: To investigate the influence of occupational aluminum exposure on cognitive function and its relationship with tri-methyl histone H3 lysine residues 4 points (H3K4me3) and brain-derived neurotrophic factor (BDNF) levels. Methods: By cluster random sampling method, a total of 235 cases of male workers selected from a Shanxi aluminum factory were recruited in the study in September 2015. Used the occupational epidemiological investigation questionnaire, which included Mini-Mental State Examination (MMSE) , Clock Drawing Test (CDT) , Digit Span Test (DST, including forward test DSFT and backward test DSBT) , Fuild Object Memory Evaluation (FOME) and Verbal Fluency Test (VFT) , to collect workers' basic information and assess their cognitive function score. Detected the concentration of aluminum in plasma by graphite furnace atomic absorption spectrometry. Workers were divided into three groups by the 25 percentile and 75 percentile of the aluminum content, such as low, middle and high aluminum concentration groups. The concentrations of H3K4me3 in lymphocyte and BDNF in plasma were determined by enzyme-linked immunosorbent assay. Results: The levels of aluminum in plasma was 134.36 (100.14, 178.96) μg/L. The scores of MMSE, DSFT, DSBT, DST of high aluminum concentration group were lower than low aluminum group (27.98±1.25 vs 28.83±1.54, 9.19±2.00 vs 10.64±2.87, 6.08±1.63 vs 7.19±3.07, 15.27±3.11 vs 17.81±4.72, all P<0.05) , the scores of CDT, FOME, VFT among three groups had no statistical significance (all P>0.05) . The expression levels of H3K4me3 and BDNF of high aluminum concentration group were lower than the low group [ (18.45±9.81) ng/μg Pro vs (23.76±9.89) ng/μg Pro, (26.07±10.18) ng/ml vs (31.66±9.24) ng/ml, all P<0.05]. Multiple correlation analysis showed that aluminum concentration were negatively correlated toH3K4me3, BDNF, MMSE, DSFT, DST, respectively (r(s)=-0.307、-0.214、-0.252、-0.197, -0.181, all P<0.01) . Conclusion: Exposure to occupational aluminum for a long time may change cognitive function, which go along with the decreasing of H3K4me3 level in lymphocyte and BDNF protein expression in plasma.

POWERDRESS and HDA9 interact and promote histone H3 deacetylation at specific genomic sites in Arabidopsis

Histone acetylation is a major epigenetic control mechanism that is tightly linked to the promotion of gene expression. Histone acetylation levels are balanced through the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Arabidopsis HDAC genes (AtHDACs) compose a large gene family, and distinct phenotypes among AtHDAC mutants reflect the functional specificity of individual AtHDACs However, the mechanisms underlying this functional diversity are largely unknown. Here, we show that POWERDRESS (PWR), a SANT (SWI3/DAD2/N-CoR/TFIII-B) domain protein, interacts with HDA9 and promotes histone H3 deacetylation, possibly by facilitating HDA9 function at target regions. The developmental phenotypes of pwr and hda9 mutants were highly similar. Three lysine residues (K9, K14, and K27) of H3 retained hyperacetylation status in both pwr and hda9 mutants. Genome-wide H3K9 and H3K14 acetylation profiling revealed elevated acetylation at largely overlapping sets of target genes in the two mutants. Highly similar gene-expression profiles in the two mutants correlated with the histone H3 acetylation status in the pwr and hda9 mutants. In addition, PWR and HDA9 modulated flowering time by repressing AGAMOUS-LIKE 19 expression through histone H3 deacetylation in the same genetic pathway. Finally, PWR was shown to physically interact with HDA9, and its SANT2 domain, which is homologous to that of subunits in animal HDAC complexes, showed specific binding affinity to acetylated histone H3. We therefore propose that PWR acts as a subunit in a complex with HDA9 to result in lysine deacetylation of histone H3 at specific genomic targets.

Boric acid-dependent decrease in regulatory histone H3 acetylation is not mutagenic in yeast.

Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations. The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast.

Genetic code expansion in stable cell lines enables encoded chromatin modification.

Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However, this approach has not been coupled to genome-wide measurements, because efficient incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl)CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-ɛ-acetyl-lysine in place of six lysine residues in histone H3 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.

Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.

GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is a histone acetyltransferase (HAT) and the catalytic subunit of several multicomponent HAT complexes that acetylate lysine residues of histone H3. Mutants in AtGCN5 display pleiotropic developmental defects including aberrant meristem function. Shoot apical meristem (SAM) maintenance is regulated by CLAVATA1 (CLV1), a receptor kinase that controls the size of the shoot and floral meristems. Upon activation through CLV3 binding, CLV1 signals to the transcription factor WUSCHEL (WUS), restricting WUS expression and thus the meristem size. We hypothesized that GCN5 and CLV1 act together to affect SAM function. Using genetic and molecular approaches, we generated and characterized clv gcn5 mutants. Surprisingly, the clv1-1 gcn5-1 double mutant exhibited constitutive ethylene responses, suggesting that GCN5 and CLV signaling act synergistically to inhibit ethylene responses in Arabidopsis. This genetic and molecular interaction was mediated by ETHYLENE INSENSITIVE 3/ EIN3-LIKE1 (EIN3/EIL1) transcription factors. Our data suggest that signals from the CLV transduction pathway reach the GCN5-containing complexes in the nucleus and alter the histone acetylation status of ethylene-responsive genes, thus translating the CLV information to transcriptional activity and uncovering a link between histone acetylation and SAM maintenance in the complex mode of ethylene signaling.

Frontiers in cardiovascular biology. Abstracts of the Third Congress of the ESC Council on Basic Cardiovascular Science. July 4-6, 2014. Barcelona, Spain.

Purpose: Interactions between chromatin-associated proteins and transcription factors play an important role in chromatin activation and gene regulation. They can be modulated by extracellular kinases, which are frequently activated by hormones and can phosphorylate chromatin-associated proteins to induce gene transcription. DPF3 is a member of the BAF (brahma-associated factor) chromatin remodeling complex and functions as an epigenetic factor to initiate gene transcription during heart and muscle development. Previous studies identified two isoforms of DPF3. DPF3b contains two PHD (plant homeo domain) fingers that recognize acetylated and methylated lysine residues of histone H3 and H4. However, very little is known of DPF3a, which contains a half-PHD finger. Here, we present the molecular mechanism of DPF3a regulation and its signaling cascade in myogenesis. Methods and Results: Using co-immunoprecipitation, we found that HEY proteins (HEY1, HEY2 and HEYL) are associated with BAF complex via the specific interaction with DPF3a. Further experiments showed that the posttranslational modification of DPF3a is essential for the interaction, which is mediated by the half-PHD finger of DPF3a. Using mass spectrometry, we identified three phosphorylation sites within DPF3a and in Tandem Affinity Purification we observed an interaction of DPF3a with all three subunits of the serine/threonine kinase CKII (casein kinase II). In vitro kinase assay showed that DPF3a is phosphorylated by CKII and inhibition of CKII by RNAi significantly reduces the DPF3a phosphorylation in vivo. DPF3a and HEY1 have common genomic targets; however, DPF3a is not capable of binding DNA directly. HEY1 knockdown in C2C12 skeletal muscle cells, combined with ChIP-qPCR demonstrated that the association of DPF3a with its targets is significantly reduced. Thus, HEY1 may represent the linker between DPF3a and its genomic targets. Interestingly, we found that the C-term of DPF3a shows high transcriptional activity in GAL4 transactivation assay and DPF3a significantly reduces HEY1 mediated repression of its targets (e.g. ANF, HEY1 and JAG1) using luciferase reporter assays. Conclusion: Our findings demonstrate an unprecedented mechanism of CKII-depending release of HEY1 from its genomic targets mediated by DPF3a and reveal a link between kinase activity and gene transcription mediated by a member of the BAF complex.

Recombinant antibodies to histone post-translational modifications

Variability in the quality of antibodies to histone post-translational modifications (PTMs) presents widely recognized hindrance in epigenetics research. Here, by using antibody engineering technologies we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and their high specificity enabled us to identify positive and negative correlations among histone PTMs.

Inhibition of PRC2 Activity by a Gain-of-Function H3 Mutation Found in Pediatric Glioblastoma

Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.