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A breakdown in host-bacteria relationships has been associated with the progression of a number of marine diseases and subsequent mortality events. For the Pacific oyster, Crassostrea gigas, summer mortality syndrome (SMS) is one of the biggest constraints to the growth of the sector and is set to expand into temperate systems as ocean temperatures rise. Currently, a lack of understanding of natural spatiotemporal dynamics of the host-bacteria relationship limits our ability to develop microbially based monitoring approaches. Here, we characterised the associated bacterial community of C. gigas, at two Irish oyster farms, unaffected by SMS, over the course of a year. We found C. gigas harboured spatiotemporally variable bacterial communities that were distinct from bacterioplankton in surrounding seawater. Whilst the majority of bacteria-oyster associations were transient and highly variable, we observed clear patterns of stability in the form of a small core consisting of six persistent amplicon sequence variants (ASVs). This core made up a disproportionately large contribution to sample abundance (34 ± 0.14%), despite representing only 0.034% of species richness across the study, and has been associated with healthy oysters in other systems. Overall, our study demonstrates the consistent features of oyster bacterial communities across spatial and temporal scales and provides an ecologically meaningful baseline to track environmental change., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Isotopic, tagging and diet studies of modern-day teleosts lacked the ability to contextualise life-history and trophic dynamics with a historical perspective, when exploitation rates were lower and climatic conditions differed. Isotopic analysis of vertebrae, the most plentiful hard-part in archaeological and museum collections, can potentially fill this data-gap. Chemical signatures of habitat and diet use during growth are retained by vertebrae during bone formation. Nonetheless, to fulfil their potential to reveal life-history and trophic dynamics, we need a better understanding of the time frame recorded by vertebrae, currently lacking due to a poor understanding of fish bone remodelling. To address this issue, the authors serially-sectioned four vertebral centra of the highly migratory Atlantic bluefin tuna (Thunnus thynnus; BFT) captured off Sardinia (Italy) and analysed their isotopic composition. They show how carbon (δ 13 C), nitrogen (δ 15 N) and sulphur (δ 34 S) isotope values can vary significantly across BFT vertebrae growth-axes, revealing patterning in dietary life histories. Further, they find that similar patterns are revealed through incremental isotopic analysis of inner and outer vertebrae centra samples from 13 archaeological BFT vertebrae dating between the 9th and13th centuries CE. The results indicate that multi-year foraging signatures are retained in vertebrae and allow for the study of life histories in both modern and paleo-environments. These novel methods can be extended across teleost taxa owing to their potential to inform management and conservation on how teleost trophic dynamics change over time and what their long-term environmental, ecological and anthropological drivers are., (© 2023 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC 50 = 79 nM) with a greater efficacy than other CDKs (IC 50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.

Atlantic bluefin tuna (Thunnus thynnus; BFT) abundance was depleted in the late 20th and early 21st century due to overfishing. Historical catch records further indicate that the abundance of BFT in the Mediterranean has been fluctuating since at least the 16th century. Here we build upon previous work on ancient DNA of BFT in the Mediterranean by comparing contemporary (2009-2012) specimens with archival (1911-1926) and archaeological (2nd century BCE-15th century CE) specimens that represent population states prior to these two major periods of exploitation, respectively. We successfully genotyped and analysed 259 contemporary and 123 historical (91 archival and 32 archaeological) specimens at 92 SNP loci that were selected for their ability to differentiate contemporary populations or their association with core biological functions. We found no evidence of genetic bottlenecks, inbreeding or population restructuring between temporal sample groups that might explain what has driven catch fluctuations since the 16th century. We also detected a putative adaptive response, involving the cytoskeletal protein synemin which may be related to muscle stress. However, these results require further investigation with more extensive genome-wide data to rule out demographic changes due to overfishing, and other natural and anthropogenic factors, in addition to elucidating the adaptive drivers related to these., (© 2021. The Author(s).)

The Italian peninsula was host to a strong history of migration processes that shaped its genomic variability since prehistoric times. During the Metal Age, Sicily and Southern Italy were the protagonists of intense trade networks and settlements along the Mediterranean. Nonetheless, ancient DNA studies in Southern Italy are, at present, still limited to prehistoric and Roman Apulia. Here, we present the first mitogenomes from a Middle Bronze Age cave burial in Calabria to address this knowledge gap. We adopted a hybridization capture approach, which enabled the recovery of one complete and one partial mitochondrial genome. Phylogenetic analysis assigned these two individuals to the H1e and H5 subhaplogroups, respectively. This preliminary phylogenetic analysis supports affinities with coeval Sicilian populations, along with Linearbandkeramik and Bell Beaker cultures maternal lineages from Central Europe and Iberia. Our work represents a starting point which contributes to the comprehension of migrations and population dynamics in Southern Italy, and highlights this knowledge gap yet to be filled by genomic studies.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis., Competing Interests: BL, RY, RH, PS, AM, AF, DF, MC, SK, CJ, AA, YH, JP No competing interests declared, (© 2021, Lowe et al.)

Dog domestication is still largely unresolved due to time-gaps in the sampling of regions. Ancient Italian canids are particularly understudied, currently represented by only a few specimens. In the present study, we sampled 27 canid remains from Northern Italy dated between the Late Pleistocene and Bronze Age to assess their genetic variability, and thus add context to dog domestication dynamics. They were targeted at four DNA fragments of the hypervariable region 1 of mitochondrial DNA. A total of 11 samples had good DNA preservation and were used for phylogenetic analyses. The dog samples were assigned to dog haplogroups A, C and D, and a Late Pleistocene wolf was set into wolf haplogroup 2. We present our data in the landscape of ancient and modern dog genetic variability, with a particular focus on the ancient Italian samples published thus far. Our results suggest there is high genetic variability within ancient Italian canids, where close relationships were evident between both a ~24,700 years old Italian canid, and Iberian and Bulgarian ancient dogs. These findings emphasize that disentangling dog domestication dynamics benefits from the analysis of specimens from Southern European regions.

Background: To mitigate potential exposure of healthcare workers (HCWs) to SARS-CoV-2 via aerosol routes, we have developed a portable hood which not only creates a barrier between HCW and patient, but also utilizes negative pressure with filtration of aerosols by a high-efficiency particulate air filter., Material and Methods: The hood has iris-port openings for access to the patient, and an opening large enough for a patient's head and upper torso. The top of the hood is a high-efficiency particulate air filter connected to a blower to apply negative pressure. We determined the aerosol penetration from outside to inside in laboratory experiments., Results: The penetration of particles from within the hood to the breathing zones of HCWs outside the hood was near 10 -4 (0.01%) in the 200-400 nm size range, and near 10 -3 (0.1%) for smaller particles. Penetration values for particles in the 500 nm-5 μm range were below 10 -2 (1%). Fluorometric analysis of deposited fluorescein particles on the personal protective equipment of an HCW revealed that negative pressure reduces particle deposition both outside and inside the hood., Conclusions: We find that negative pressure hoods can be effective controls to mitigate aerosol exposure to HCWs, while simultaneously allowing access to patients., (Copyright © 2020 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Canned tuna is one of the most widely traded seafood products internationally and is of growing demand. There is an increasing concern over the vulnerability of canned tuna supply chains to species mislabelling and fraud. Extensive processing conditions in canning operations can lead to the degradation and fragmentation of DNA, complicating product traceability. We here employed a forensically validated DNA barcoding tool (cytochrome b partial sequences) to assess the effects of canning processes on DNA degradation and the identification of four tropical tuna species (yellowfin, bigeye, skipjack and longtail tuna) collected on a global scale, along their commercial chains. Each species was studied under five different canning processes i.e., freezing, defrosting, cooking, and canning in oil and brine, in order to investigate how these affect DNA-based species identification and traceability. The highest percentage of nucleotide substitutions were observed after brine-canning operations and were greatest for yellowfin and skipjack tuna. Overall, we found that DNA degradation significantly increased along the tuna canning process for most specimens. Consequently, most of the specimens canned in oil or brine were misidentified due to the high rate of nucleotide substitution in diagnostic sequences.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo., Competing Interests: The author(s) declare no competing interests.

The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56., (Published by Oxford University Press on behalf of Nucleic Acids Research 2019.)

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer., (© 2019 Sidoli et al.; Published by Cold Spring Harbor Laboratory Press.)

As a result of ocean warming, the species composition of the Arctic seas has begun to shift in a boreal direction. One ecosystem prone to fauna shifts is the Northeast Greenland shelf. The dispersal route taken by boreal fauna to this area is, however, not known. This knowledge is essential to predict to what extent boreal biota will colonise Arctic habitats. Using population genetics, we show that Atlantic cod (Gadus morhua), beaked redfish (Sebastes mentella), and deep-sea shrimp (Pandalus borealis) recently found on the Northeast Greenland shelf originate from the Barents Sea, and suggest that pelagic offspring were dispersed via advection across the Fram Strait. Our results indicate that boreal invasions of Arctic habitats can be driven by advection, and that the fauna of the Barents Sea can project into adjacent habitats with the potential to colonise putatively isolated Arctic ecosystems such as Northeast Greenland.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca 2+ -dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca 2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Purpose: The role of cholesterol biosynthesis in hedgehog pathway activity and progression of hedgehog pathway medulloblastoma (Hh-MB) were examined in vivo Statins, commonly used cholesterol-lowering agents, were utilized to validate cholesterol biosynthesis as a therapeutic target for Hh-MB. Experimental Design: Bioinformatic analysis was performed to evaluate the association between cholesterol biosynthesis with hedgehog group medulloblastoma in human biospecimens. Alterations in hedgehog signaling were evaluated in medulloblastoma cells after inhibition of cholesterol biosynthesis. The progression of endogenous medulloblastoma in mice was examined after genetic blockage of cholesterol biosynthesis in tumor cells. Statins alone, or in combination with vismodegib (an FDA-approved Smoothened antagonist), were utilized to inhibit medulloblastoma growth in vivo Results: Cholesterol biosynthesis was markedly enhanced in Hh-MB from both humans and mice. Inhibition of cholesterol biosynthesis dramatically decreased Hh pathway activity and reduced proliferation of medulloblastoma cells. Statins effectively inhibited medulloblastoma growth in vivo and functioned synergistically in combination with vismodegib. Conclusions: Cholesterol biosynthesis is required for Smoothened activity in the hedgehog pathway, and it is indispensable for the growth of Hh-MB. Targeting cholesterol biosynthesis represents a promising strategy for treatment of Hh-MB. Clin Cancer Res; 24(6); 1375-88. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Objective: IL-35 (interleukin-35) is an anti-inflammatory cytokine, which inhibits immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. It remains unknown whether atherogenic stimuli induce IL-35 and whether IL-35 inhibits atherogenic lipid-induced endothelial cell (EC) activation and atherosclerosis. EC activation induced by hyperlipidemia stimuli, including lysophosphatidylcholine is considered as an initiation step for monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the roles and mechanisms of IL-35 in suppressing lysophosphatidylcholine-induced EC activation., Approach and Results: Using microarray and ELISA, we found that IL-35 and its receptor are significantly induced during early atherosclerosis in the aortas and plasma of ApoE (apolipoprotein E) knockout mice-an atherosclerotic mouse model-and in the plasma of hypercholesterolemic patients. In addition, we found that IL-35 suppresses lysophosphatidylcholine-induced monocyte adhesion to human aortic ECs. Furthermore, our RNA-sequencing analysis shows that IL-35 selectively inhibits lysophosphatidylcholine-induced EC activation-related genes, such as ICAM-1 (intercellular adhesion molecule-1). Mechanistically, using flow cytometry, mass spectrometry, electron spin resonance analyses, and chromatin immunoprecipitation-sequencing analyses, we found that IL-35 blocks lysophosphatidylcholine-induced mitochondrial reactive oxygen species, which are required for the induction of site-specific H3K14 (histone 3 lysine 14) acetylation, increased binding of proinflammatory transcription factor AP-1 in the promoter of ICAM-1, and induction of ICAM-1 transcription in human aortic EC. Finally, IL-35 cytokine therapy suppresses atherosclerotic lesion development in ApoE knockout mice., Conclusions: IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation., (© 2018 American Heart Association, Inc.)

Elevated plasma total homocysteine (tHcy) is associated with a number of human diseases including coronary artery disease, stroke, osteoporosis and dementia. It is highly correlated with intracellular S-adenosylhomocysteine (SAH). Since SAH is a strong inhibitor of methyl-transfer reactions involving the methyl-donor S-adenosylmethionine (SAM), elevation in SAH could be an explanation for the wide association of tHcy and human disease. Here, we have created a transgenic mouse ( Tg-hAHCY ) that expresses human S-adenosylhomocysteine hydrolase ( AHCY ) from a zinc-inducible promoter in the liver and kidney. Protein analysis shows that human AHCY is expressed well in both liver and kidney, but elevated AHCY enzyme activity (131% increase) is only detected in the kidney due to the high levels of endogenous mouse AHCY expression in liver. Tg-hAHCY mice were crossed with mice lacking cystathionine β-synthase activity ( Tg-I278T Cbs -/- ) to explore the effect to AHCY overexpression in the context of elevated serum tHcy and elevated tissue SAM and SAH. Overexpression of AHCY had no significant effect on the phenotypes of Tg-I278T Cbs -/- mice or any effect on the steady state concentrations of methionine, total homocysteine, SAM, SAH, and SAM/SAH ratio in the liver and kidney. Furthermore, enhanced AHCY activity did not lower serum and tissue tHcy or methionine levels. Our data suggests that enhancing AHCY activity does not alter the distribution of methionine recycling metabolites, even when they are greatly elevated by Cbs mutations.

Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas, and meningiomas. Mutational inactivation of the NF2 gene encoding the protein Merlin is found in most sporadic and inherited schwannomas, but the molecular mechanisms underlying neoplastic changes in schwannoma cells remain unclear. We report here that Nf2-deficient cells display elevated expression levels of key enzymes involved in lipogenesis and that this upregulation is caused by increased activity of Torc1. Inhibition or knockdown of fatty acid synthase (FASN), the enzyme that catalyzes the formation of palmitic acid from malonyl-CoA, drove NF2 -deficient cells into apoptosis. Treatment of NF2 -mutant cells with agents that inhibit the production of malonyl-CoA reduced their sensitivity to FASN inhibitors. Collectively, these results suggest that the altered lipid metabolism found in NF2 -mutant cells renders them sensitive to elevated levels of malonyl-CoA, as occurs following blockade of FASN, suggesting new targeted strategies in the treatment of NF2 -deficient tumors. Cancer Res; 77(18); 5026-38. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Metastatic melanoma cells commonly acquire resistance to BRAF V600E inhibitors (BRAFi). In this study, we identified serine biosynthesis as a critical mechanism of resistance. Proteomic assays revealed differential protein expression of serine biosynthetic enzymes PHGDH, PSPH, and PSAT1 following vemurafenib (BRAFi) treatment in sensitive versus acquired resistant melanoma cells. Ablation of PHGDH via siRNA sensitized acquired resistant cells to vemurafenib. Inhibiting the folate cycle, directly downstream of serine synthesis, with methotrexate also displayed similar sensitization. Using the DNA-damaging drug gemcitabine, we show that gemcitabine pretreatment sensitized resistant melanoma cells to BRAFis vemurafenib and dabrafenib. We extended our findings to BRAF WT tumor cell lines that are intrinsically resistant to vemurafenib and dabrafenib. Pretreatment of pancreatic cancer and non-small cell lung cancer cell lines with sublethal doses of 50 and 5 nmol/L of gemcitabine, respectively, enhanced killing by both vemurafenib and dabrafenib. The novel aspects of this study are the direct identification of serine biosynthesis as a critical mechanism of BRAF V600E inhibitor resistance and the first successful example of using gemcitabine + BRAFis in combination to kill previously drug-resistant cancer cells, creating the translational potential of pretreatment with gemcitabine prior to BRAFi treatment of tumor cells to reverse resistance within the mutational profile and the WT. Mol Cancer Ther; 16(8); 1596-609. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [ N -(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation., Competing Interests: Notes The authors declare no competing financial interest.

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency., Competing Interests: Authors have no conflicts of interest to declare., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.

Objective: Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis., Approach and Results: Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0, and 18:1 induced mtROS in primary human aortic ECs, independently of the activities of nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry-mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of activator protein-1 and inducing intercellular adhesion molecule-1 gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in apolipoprotein E knockout mice using intravital microscopy and flow cytometry methods., Conclusions: ATP synthesis-uncoupled, but proton leak-coupled, mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases., (© 2016 American Heart Association, Inc.)

Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling.

Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.

Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer., (©2015 American Association for Cancer Research.)

Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions., (Copyright © 2015. Published by Elsevier B.V.)

We have a limited understanding of the site specificity of multi-subunit lysine acetyltransferase (KAT) complexes for histone-based substrates, especially in regards to the different complexes formed during nucleosome assembly. Histone complexes could be a major factor in determining the acetylation specificity of KATs. In the present study, we utilized a label-free quantitative MS-based method to determine the site specificity of acetylation catalysed by Piccolo NuA4 on (H3/H4)2 tetramer, tetramer bound DNA (tetrasome) and nucleosome core particle (NCP). Our results show that Piccolo NuA4 can acetylate multiple lysine residues on these three histone complexes, of which NCP is the most favourable, (H3/H4)2 tetramer is the second and tetrasome is the least favourable substrate for Piccolo NuA4 acetylation. Although Piccolo NuA4 preferentially acetylates histone H4 (H4K12), the site specificity of the enzyme is altered with different histone complex substrates. Our results show that before nucleosome assembly is complete, H3K14 specificity is almost equal to that of H4K12 and DNA-histone interactions suppress the acetylation ability of Piccolo NuA4. These data suggest that the H2A/H2B dimer could play a critical role in the increase in acetylation specificity of Piccolo NuA4 for NCP. This demonstrates that histone complex formation can alter the acetylation preference of Piccolo NuA4. Such findings provide valuable insight into regulating Piccolo NuA4 specificity by modulating chromatin dynamics and in turn manipulating gene expression., (© 2015 Authors; published by Portland Press Limited.)

In Saccharomyces cerevisiae, Rtt109, a lysine acetyltransferase (KAT), associates with a histone chaperone, either Vps75 or Asf1. It has been proposed that these chaperones alter the selectivity of Rtt109 or which residues it preferentially acetylates. In the present study, we utilized a label-free quantitative mass spectrometry-based method to determine the steady-state kinetic parameters of acetylation catalyzed by Rtt109-Vps75 on H3 monomer, H3/H4 tetramer, and H3/H4-Asf1 complex. These results show that among these histone conformations, only H3K9 and H3K23 are significantly acetylated under steady-state conditions and that Asf1 promotes H3/H4 acetylation by Rtt109-Vps75. Asf1 equally increases the Rtt109-Vps75 specificity for both of these residues with a maximum stoichiometry of 1:1 (Asf1 to H3/H4), but does not alter the selectivity between these two residues. These data suggest that the H3/H4-Asf1 complex is a substrate for Rtt109-Vps75 without altering selectivity between residues. The deletion of either Rtt109 or Asf1 in vivo results in the same reduction of H3K9 acetylation, suggesting that Asf1 is required for efficient H3K9 acetylation both in vitro and in vivo. Furthermore, we found that the acetylation preference of Rtt109-Vps75 could be directed to H3K56 when those histones already possess modifications, such as those found on histones purified from chicken erythrocytes. Taken together, Vps75 and Asf1 both enhance Rtt109 acetylation for H3/H4, although via different mechanisms, but have little impact on the residue selectivity. Importantly, these results provide evidence that histone chaperones can work together via interactions with either the enzyme or the substrate to more efficiently acetylate histones.

Determining how histone acetylation is regulated is vital for treating the many diseases associated with its misregulation, including heart disease, neurological disorders, and cancer. We have previously reported that acetyl-CoA levels alter p300 histone acetylation in a site-specific manner in vitro. Here, we further investigate how changing acetyl-CoA concentrations alter the histone acetylation pattern by altering p300 specificity. Interestingly, these changes are not a simple global change in acetylation, but rather site specific changes, whereby acetylation at some sites increase while others decrease. We also demonstrate how the p300 inhibitor C646 can pharmacologically alter p300 histone acetylation patterns in vitro and in cells. This study provides insight into the mechanisms regulating p300 residue specificity, a potential means for altering p300 dependent histone acetylation, and an investigation into altering histone acetylation patterns in cells.

Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Recent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP. The majority of these mutations reduce or eliminate the catalytic HAT activity. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300ΔC-1087), whose expression is essential for cell proliferation., Methods: Using results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300ΔC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300ΔC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300ΔC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300., Results: We show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300ΔC-820), but no wild-type p300. The p300ΔC-820 protein has a wild-type ability to localize to subnuclear "speckles," but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300ΔC-820 in SUDHL2 cells reduced their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300ΔC-1087 resulted in increased expression of mRNA and protein for REL target genes A20 and IκBα, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18., Conclusions: The high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.

Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.

Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.

Communication barriers often result in exclusion of children and youth with disabilities from activities and social settings that are essential to their psychosocial development. In particular, difficulties in describing their experiences of activities and social settings hinder our understanding of the factors that promote inclusion and participation of this group of individuals. To address this specific communication challenge, we examined the feasibility of developing a language-free measure of experience in youth with severe physical disabilities. To do this, we used the activity of the peripheral nervous system to detect patterns of psychological arousal associated with activities requiring different patterns of cognitive/affective and interpersonal involvement (activity engagement). We demonstrated that these signals can differentiate among patterns of arousal associated with these activities with high accuracy (two levels: 81%, three levels: 74%). These results demonstrate the potential for development of a real-time, motor- and language-free measure for describing the experiences of children and youth with disabilities.

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A-H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3-H4)(2) tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)(2) tetramer removal. We estimate that the open intermediate state is populated at 0.2-3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.

Chromatin is a highly regulated, modular nucleoprotein complex that is central to many processes in eukaryotes. The organization of DNA into nucleosomes and higher-order structures has profound implications for DNA accessibility. Alternative structural states of the nucleosome, and the thermodynamic parameters governing its assembly and disassembly, need to be considered in order to understand how access to nucleosomal DNA is regulated. In this review, we provide a brief historical account of how the overriding perception regarding aspects of nucleosome structure has changed over the past thirty years. We discuss recent technical advances regarding nucleosome structure and its physical characterization and review the evidence for alternative nucleosome conformations and their implications for nucleosome and chromatin dynamics.

The repeating structural unit of eukaryotic chromatin, the nucleosome, is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. These proteins form an octamer around which 147bp of DNA is wrapped in 1.65 superhelical turns (Luger et al., 1997). The nucleosome represents a major obstacle for any protein seeking access to the DNA. Several strategies have evolved to regulate access to nucleosomal DNA, such as posttranslational modification of histones and histone variants, ATP-dependent chromatin remodeling machines, and histone chaperones. It is likely that most if not all of these mechanisms directly impact the thermodynamics of the nucleosome. The DNA sequence itself may also impact its own inherent accessibility through modulating nucleosome positioning and/or thermodynamics. However, these working hypotheses could not be tested directly because no assays to measure nucleosome stability under physiological conditions were available. Attempts to determine the stability of nucleosomes have been hampered by the fact that the nucleosomes do not assemble in vitro under physiological conditions, but will only form nucleosomes through titration from high (2M) to low (>0.3M) ionic strength. We have developed a coupled equilibrium approach using the histone chaperone Nap1 to measure nucleosome thermodynamics under physiological conditions. This method will be useful for examining the impact of DNA sequence, histone variants, and posttranslational modifications on nucleosome thermodynamics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The organization of the eukaryotic genome into nucleosomes dramatically affects the regulation of gene expression. The delicate balance between transcription and DNA compaction relies heavily on nucleosome dynamics. Surprisingly, little is known about the free energy required to assemble these large macromolecular complexes and maintain them under physiological conditions. Here, we describe the thermodynamic parameters that drive nucleosome formation in vitro. To demonstrate the versatility of our approach, we test the effect of DNA sequence and H3K56 acetylation on nucleosome thermodynamics. Furthermore, our studies reveal the mechanism of action of the histone chaperone nucleosome assembly protein 1 (Nap1). We present evidence for a paradigm in which nucleosome assembly requires the elimination of competing, nonnucleosomal histone-DNA interactions by Nap1. This observation is confirmed in vivo, wherein deletion of the NAP1 gene in yeast results in a significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression., ((c) 2010 Elsevier Inc. All rights reserved.)

Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5' end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn(2+) bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 A. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn(2+) paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with , Zn(2+), and confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.

The flavivirus 2'-O-nucleoside N-terminal RNA methyltransferase (MTase) enzyme is responsible for methylating the viral RNA cap structure. To increase our understanding of the mechanism of viral RNA cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (DEN) and yellow fever (YF) virus MTase enzymes. We solved an improved 2.1 A resolution crystal structure of DEN2 Mtase, new 1.5 A resolution crystal structures of the YF virus MTase domain in apo form, and a new 1.45 A structure in complex with guanosine triphosphate and RNA cap analog. Our structures clarify the previously reported DEN MTase structure, suggest novel protein-cap interactions, and provide a detailed view of guanine specificity. Furthermore, the structures of the DEN and YF proteins are essentially identical, indicating a large degree of structural conservation amongst the flavivirus MTases. Guanosine triphosphate analog competition assays and mutagenesis analysis, performed to analyze the biochemical characteristics of cap binding, determined that the major interaction points are (i) guanine ring via pi-pi stacking with Phe24, N1 hydrogen interaction with the Leu19 backbone carbonyl via a water bridge, and C2 amine interaction with Leu16 and Leu19 backbone carbonyls; (ii) ribose 2' hydroxyl interaction with Lys13 and Asn17; and (iii) alpha-phosphate interactions with Lys28 and Ser215. Based on our mutational and analog studies, the guanine ring and alpha-phosphate interactions provide most of the energy for cap binding, while the combination of the water bridge between the guanine N1 and Leu19 carbonyl and the hydrogen bonds between the C2 amine and Leu16/Leu19 carbonyl groups provide for specific guanine recognition. A detailed model of how the flavivirus MTase protein binds RNA cap structures is presented.

The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nm affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4)(2) tetramer binds DNA with higher affinity than it binds yNap1.

Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone 3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. Here we show that both Rtt109 and Vps75 bind histones with high affinity, but only the complex is efficient for catalysis. The C-terminal acidic domain of Vps75 contributes to activation of Rtt109 and is necessary for in vivo functionality of Vps75, but it is not required for interaction with either Rtt109 or histones. We demonstrate that Vps75 is a structural homolog of yeast Nap1 by solving its crystal structure. Nap1 and Vps75 interact with histones and Rtt109 with comparable affinities. However, only Vps75 stimulates Rtt109 enzymatic activity. Our data highlight the functional specificity of Vps75 in Rtt109 activation.