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Purpose Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that modulates intracellular transport and protein quality control. Inhibition of HDAC6 deacetylase activity has shown beneficial effects in disease models, including Alzheimer’s disease and amyotrophic lateral sclerosis. This first-in-human positron emission tomography (PET) study evaluated the brain binding of [18F]EKZ-001 ([18F]Bavarostat), a radiotracer selective for HDAC6, in healthy adult subjects. Methods Biodistribution and radiation dosimetry studies were performed in four healthy subjects (2M/2F, 23.5 ± 2.4 years) using sequential whole-body PET/CT. The most appropriate kinetic model to quantify brain uptake was determined in 12 healthy subjects (6M/6F, 57.6 ± 3.7 years) from 120-min dynamic PET/MR scans using a radiometabolite-corrected arterial plasma input function. Four subjects underwent retest scans (2M/2F, 57.3 ± 5.6 years) with a 1-day interscan interval to determine test-retest variability (TRV). Regional volume of distribution (VT) was calculated using one-tissue and two-tissue compartment models (1-2TCM) and Logan graphical analysis (LGA), with time-stability assessed. VT differences between males and females were evaluated using volume of interest and whole-brain voxel-wise approaches. Results The effective dose was 39.1 ± 7.0 μSv/MBq. Based on the Akaike information criterion, 2TCM was the preferred model compared to 1TCM. Regional LGA VT were in agreement with 2TCM VT, however demonstrated a lower absolute TRV of 7.7 ± 4.9%. Regional VT values were relatively homogeneous with highest values in the hippocampus and entorhinal cortex. Reduction of acquisition time was achieved with a 0 to 60-min scan followed by a 90 to 120-min scan. Males demonstrated significantly higher VT than females in the majority of cortical and subcortical brain regions. No relevant radiotracer related adverse events were reported. Conclusion [18F]EKZ-001 is safe and appropriate for quantifying HDAC6 expression in the human brain with Logan graphical analysis as the preferred quantitative approach. Males showed higher HDAC6 expression across the brain compared to females.

The etiology of bipolar disorder (BD) is unknown and the neurobiological underpinnings are not fully understood. Both genetic and environmental factors contribute to the risk of BD, which may be linked through epigenetic mechanisms, including those regulated by histone deacetylase (HDAC) enzymes. This study measures in vivo HDAC expression in individuals with BD for the first time using the HDAC-specific radiotracer [11C]Martinostat. Eleven participants with BD and 11 age- and sex-matched control participants (CON) completed a simultaneous magnetic resonance – positron emission tomography (MR-PET) scan with [11C]Martinostat. Lower [11C]Martinostat uptake was found in the right amygdala of BD compared to CON. We assessed uptake in the dorsolateral prefrontal cortex (DLPFC) to compare previous findings of lower uptake in the DLPFC in schizophrenia and found no group differences in BD. Exploratory whole-brain voxelwise analysis showed lower [11C]Martinostat uptake in the bilateral thalamus, orbitofrontal cortex, right hippocampus, and right amygdala in BD compared to CON. Furthermore, regional [11C]Martinostat uptake was associated with emotion regulation in BD in fronto-limbic areas, which aligns with findings from previous structural, functional, and molecular neuroimaging studies in BD. Regional [11C]Martinostat uptake was associated with attention in BD in fronto-parietal and temporal regions. These findings indicate a potential role of HDACs in BD pathophysiology. In particular, HDAC expression levels may modulate attention and emotion regulation, which represent two core clinical features of BD.

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic enzyme involved in diverse cellular processes such as intracellular transport and protein quality control. Inhibition of HDAC6 can alleviate defects in cell and rodent models of certain diseases, particularly neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis. However, while HDAC6 represents a potentially powerful therapeutic target, development of effective brain-penetrant HDAC6 inhibitors remains challenging. Recently, [18F]EKZ-001 ([18F]Bavarostat), a brain-penetrant positron emission tomography (PET) radioligand with high affinity and selectivity toward HDAC6, was developed and evaluated preclinically for its ability to bind HDAC6. Herein, we describe the efficient and robust fully automated current Good Manufacturing Practices (cGMP) compliant production method. [18F]EKZ-001 quantification methods were validated in nonhuman primates (NHP) using full kinetic modeling, and [18F]EKZ-001 PET was applied to compare dose-occupancy relationships between two HDAC6 inhibitors, EKZ-317 and ACY-775. [18F]EKZ-001 is cGMP produced with an average decay-corrected radiochemical yield of 14% and an average molar activity of 204 GBq/μmol. We demonstrate that a two-tissue compartmental model and Logan graphical analysis are appropriate for [18F]EKZ-001 PET quantification in NHP brain. Blocking studies show that the novel compound EKZ-317 achieves higher target occupancy than ACY-775. This work supports the translation of [18F]EKZ-001 PET for first-in-human studies. ispartof: ACS CHEMICAL NEUROSCIENCE vol:11 issue:7 pages:1093-1101 ispartof: location:United States status: published

Age- and sex-related alterations in gene transcription have been demonstrated, however the underlying mechanisms are unresolved. Neuroepigenetic pathways regulate gene transcription in the brain. Here, we measure in vivo expression of the epigenetic enzymes, histone deacetylases (HDACs), across healthy human aging and between sexes using [11C]Martinostat positron emission tomography (PET) neuroimaging (n = 41). Relative HDAC expression increases with age in cerebral white matter, and correlates with age-associated disruptions in white matter microstructure. A post mortem study confirmed that HDAC1 and HDAC2 paralogs are elevated in white matter tissue from elderly donors. There are also sex-specific in vivo HDAC expression differences in brain regions associated with emotion and memory, including the amygdala and hippocampus. Hippocampus and white matter HDAC expression negatively correlates with emotion regulation skills (n = 23). Age and sex are associated with HDAC expression in vivo, which could drive age- and sex-related transcriptional changes and impact human behavior., Gene transcription is known to vary with age and sex, although the underlying mechanisms are unresolved. Here, the authors show that epigenetic enzymes known as HDACs, which regulate gene transcription, are increasingly expressed with age in the living human brain, with sex differences also observed.

BACKGROUND. Patients with schizophrenia (SCZ) experience chronic cognitive deficits. Histone deacetylases (HDACs) are enzymes that regulate cognitive circuitry; however, the role of HDACs in cognitive disorders, including SCZ, remains unknown in humans. We previously determined that HDAC2 mRNA levels were lower in dorsolateral prefrontal cortex (DLPFC) tissue from donors with SCZ compared with controls. Here we investigated the relationship between in vivo HDAC expression and cognitive impairment in patients with SCZ and matched healthy controls using [(11)C]Martinostat positron emission tomography (PET). METHODS. In a case-control study, relative [((11))C]Martinostat uptake was compared between 14 patients with SCZ or schizoaffective disorder (SCZ/SAD) and 17 controls using hypothesis-driven region-of-interest analysis and unbiased whole brain voxel-wise approaches. Clinical measures, including the MATRICS consensus cognitive battery, were administered. RESULTS. Relative HDAC expression was lower in the DLPFC of patients with SCZ/SAD compared with controls, and HDAC expression positively correlated with cognitive performance scores across groups. Patients with SCZ/SAD also showed lower relative HDAC expression in the dorsomedial prefrontal cortex and orbitofrontal gyrus, and higher relative HDAC expression in the cerebral white matter, pons, and cerebellum compared with controls. CONCLUSIONS. These findings provide in vivo evidence of HDAC dysregulation in patients with SCZ and suggest that altered HDAC expression may impact cognitive function in humans. FUNDING. National Institute of Mental Health (NIMH), Brain and Behavior Foundation, Massachusetts General Hospital (MGH), Athinoula A. Martinos Center for Biomedical Imaging, National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH Shared Instrumentation Grant Program.

RationaleHistone deacetylases (HDACs) play a central role in cardiac hypertrophy and fibrosis in preclinical models. However, their impact in the human heart remains unknown.ObjectiveWe aimed to image HDAC expression in the human heart in vivo with PET-MR (positron emission tomography and magnetic resonance) using [11C]Martinostat, a novel radiotracer targeted to class I HDACs. We further aimed to compare HDAC expression in the heart with its expression in skeletal muscle and brown/white adipose tissue (BAT/WAT).Methods and ResultsThe specificity and selectivity of [11C]Martinostat binding in the heart was assessed in non-human primates (n=2) by in vivo blocking studies and with an ex vivo cellular thermal shift assay (CETSA) of HDAC paralog stabilization by Martinostat. PET-MR imaging of [11C]Martinostat was performed in healthy volunteers (n=6) for 60 minutes to obtain time-activity curves of probe uptake and kinetics. qPCR of class I HDACs was performed in specimens of BAT obtained from patients (n=7) undergoing abdominal surgery and in specimens of human subcutaneous WAT (n=7). CETSA and the blocking studies demonstrated that Martinostat was specific for class I HDACs in the heart. HDAC density, measured by standardized uptake values of [11C]Martinostat, was 8 times higher in the myocardium than skeletal muscle (4.4 ± 0.6 vs. 0.54 ± 0.29, pConclusionsClass I HDAC expression in the human heart can be imaged in vivo and is dramatically higher than any other peripheral tissue, including skeletal muscle. The high levels of HDAC in the myocardium and BAT suggest that epigenetic regulation plays an important role in tissues with high energetic demands and metabolic plasticity.

Purpose Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that modulates intracellular transport and protein quality control. Inhibition of HDAC6 deacetylase activity has shown beneficial effects in disease models, including Alzheimer’s disease and amyotrophic lateral sclerosis. This first-in-human positron emission tomography (PET) study evaluated the brain binding of [18F]EKZ-001 ([18F]Bavarostat), a radiotracer selective for HDAC6, in healthy adult subjects. Methods Biodistribution and radiation dosimetry studies were performed in four healthy subjects (2M/2F, 23.5 ± 2.4 years) using sequential whole-body PET/CT. The most appropriate kinetic model to quantify brain uptake was determined in 12 healthy subjects (6M/6F, 57.6 ± 3.7 years) from 120-min dynamic PET/MR scans using a radiometabolite-corrected arterial plasma input function. Four subjects underwent retest scans (2M/2F, 57.3 ± 5.6 years) with a 1-day interscan interval to determine test-retest variability (TRV). Regional volume of distribution (VT) was calculated using one-tissue and two-tissue compartment models (1-2TCM) and Logan graphical analysis (LGA), with time-stability assessed. VT differences between males and females were evaluated using volume of interest and whole-brain voxel-wise approaches. Results The effective dose was 39.1 ± 7.0 μSv/MBq. Based on the Akaike information criterion, 2TCM was the preferred model compared to 1TCM. Regional LGA VT were in agreement with 2TCM VT, however demonstrated a lower absolute TRV of 7.7 ± 4.9%. Regional VT values were relatively homogeneous with highest values in the hippocampus and entorhinal cortex. Reduction of acquisition time was achieved with a 0 to 60-min scan followed by a 90 to 120-min scan. Males demonstrated significantly higher VT than females in the majority of cortical and subcortical brain regions. No relevant radiotracer related adverse events were reported. Conclusion [18F]EKZ-001 is safe and appropriate for quantifying HDAC6 expression in the human brain with Logan graphical analysis as the preferred quantitative approach. Males showed higher HDAC6 expression across the brain compared to females.

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic enzyme involved in diverse cellular processes such as intracellular transport and protein quality control. Inhibition of HDAC6 can alleviate defects in cell and rodent models of certain diseases, particularly neurodegenerative disorders, including Alzheimer’s disease and amyotrophic lateral sclerosis. However, while HDAC6 represents a potentially powerful therapeutic target, development of effective brain-penetrant HDAC6 inhibitors remains challenging. Recently [(18)F]EKZ-001 ([(18)F]Bavarostat), a brain-penetrant positron emission tomography (PET) radioligand with high affinity and selectivity towards HDAC6, was developed and evaluated preclinically for its ability to bind HDAC6. Herein, we describe the efficient and robust fully automated current good manufacturing practices (cGMP) compliant production method. [(18)F]EKZ-001 quantification methods were validated in non-human primates (NHP) using full kinetic modelling and [(18)F]EKZ-001 PET was applied to compare dose-occupancy relationships between two HDAC6 inhibitors, EKZ-317 and ACY-775. [(18)F]EKZ-001 is cGMP produced with an average decay-corrected radiochemical yield of 14 % and an average molar activity of 204 GBq/μmol. We demonstrate that a two-tissue compartmental model and Logan graphical analysis are appropriate for [(18)F]EKZ-001 PET quantification in NHP brain. Blocking studies show that the novel compound, EKZ-317, achieves higher target occupancy than ACY-775. This work supports the translation of [(18)F]EKZ-001 PET for first-in-human studies.

Postmortem brain studies support dysregulated expression of the histone deacetylase enzymes, HDAC1 and HDAC2, as a central feature in diseases including schizophrenia, bipolar disorder, and depression. Our objective was to investigate HDAC expression in a large postmortem sample set representing healthy and disease brains. We used >700 well-characterized samples from patients diagnosed with schizophrenia (n = 175), major depressive disorder (n = 135), and bipolar disorder (n = 61) to measure HDAC1 and HDAC2 transcript levels by quantitative real-time PCR in dorsolateral prefrontal cortex (DLPFC) and caudate compared to control samples. HDAC expression was calculated relative to the geometric mean of β-2-microglobulin, β-glucuronidase, and β-actin. In adult-age DLPFC, HDAC2 was decreased by 34% in schizophrenia samples compared to controls (p < 10–4). HDAC2 was significantly upregulated in major depressive disorder samples by 17% versus controls (p = 0.002). Neither smoking history nor therapeutic drugs impacted HDAC2 levels and no HDAC1 patient-control differences were observed. In caudate, HDAC levels were unchanged between patient and control groups. In control DLPFC, age fetal week 14 to 97 years (n = 326), both HDAC1 and HDAC2 levels sharply declined around birth and stabilized thereafter. Using by far the largest postmortem sample set on this topic, our major finding (decreased HDAC2 transcript) showed notable specificity in disease (schizophrenia but not major depressive disorder), HDAC subtype (HDAC2 but not HDAC1) and brain region (DLPFC but not caudate). These differences shape understanding of regional components of neural circuitry in the diseased brain and set a benchmark to quantify HDAC density and distribution using in vivo neuroimaging tools.

Introduction There is an unmet need for mechanism-based biomarkers and effective disease modifying treatments in amyotrophic lateral sclerosis (ALS). Previous findings have provided evidence that histone deacetylases (HDAC) are altered in ALS, providing a rationale for testing HDAC inhibitors as a therapeutic option. Methods We measured class I and II HDAC protein and transcript levels together with acetylation levels of downstream substrates by using Western blotting in postmortem tissue of ALS and controls. [11 C]Martinostat, a novel HDAC positron emission tomography ligand, was also used to assess in vivo brain HDAC alterations in patients with ALS and healthy controls (HC). Results There was no significant difference in HDAC levels between patients with ALS and controls as measured by Western blotting and reverse-transcription quantitative polymerase chain reaction. Similarly, no differences were detected in [11 C]Martinostat-positron emission tomography uptake in ALS participants compared with HCs. Discussion These findings provide evidence that alterations in HDAC isoforms are not a dominant pathological feature at the bulk tissue level in ALS.

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5'-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156.

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.

Various sizes of gold nano colloidal particles ranging from 5 nm to 100 nm of size were encapsulated in a silica based sol–gel, and these surfaces were exposed to a pH 1 acid solution. This enabled us to observe the process of solvent intrusion and interaction with gold colloids by the absorption spectrum as a function of time. The rate was analyzed by a single exponential analytical function, and the maximum rate was found for gold colloid of 15 nm size. The least acid interaction and colour change was observed for the size of 60 nm. It was speculated that the surface of these gold colloids were homogeneously covered by the sodium tetra-borate buffer which insulated silica gel layer, thus avoiding direct contact of the acid with the surface of the gold colloid. This study confirmed that the nano scale dopant size affects the rate of solvent penetration into a sol–gel cavity.

Epigenetic dysfunction is implicated in many neurological and psychiatric diseases, including Alzheimer’s disease and schizophrenia. Consequently, histone deacetylases (HDACs) are being aggressively pursued as therapeutic targets. However, a fundamental knowledge gap exists regarding the expression and distribution of HDACs in healthy individuals for comparison to disease states. Here, we report the first-in-human evaluation of neuroepigenetic regulation in vivo. Using positron emission tomography (PET) with [11C]Martinostat, an imaging probe selective for class I HDACs (isoforms 1–3), we found HDAC expression is higher in cortical gray matter than white matter with strikingly conserved regional distribution patterns within and between healthy individuals. Among gray matter regions, HDAC expression is lowest in the hippocampus and amygdala. Through biochemical profiling of postmortem human brain tissue, we confirmed [11C]Martinostat selectively binds HDAC isoforms 1–3, the HDAC subtypes most implicated in regulating neuroplasticity and cognitive function. To relate the PET imaging signal to the epigenetic regulation of gene transcription, we measured mRNA expression changes elicited by Martinostat in human stem cell-derived neural progenitor cells. We demonstrate that genes closely associated with synaptic plasticity, including BDNF (brain derived neurotrophic factor) and SYP (synaptophysin), as well as genes implicated in neurodegeneration, including GRN (progranulin), were markedly increased at the transcript level in concert with increased acetylation at both histone H3 lysine 9 and histone H4 lysine 12. This study provides the first quantification of HDAC expression in the living human brain, and provides the foundation for gaining unprecedented in vivo epigenetic information in the healthy and diseased human brain.

Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs. DOI: http://dx.doi.org/10.7554/eLife.02996.001, eLife digest Inside the nucleus of a cell, the DNA is wound around histone proteins. This forms a structure called chromatin that allows the long DNA strands to fit inside the cell. Variations in chromatin structure also help the cell to control the functional properties of DNA. For example, a large proportion of chromatin in the cell is in the form of heterochromatin, which is very densely packed, and is associated with many roles such as gene silencing and keeping DNA intact during reproduction. Many animals and plants have two copies of each DNA molecule: one inherited from the mother, and one from the father of the organism. Reproductive cells undergo a process called recombination when they form, where the matching copies of each DNA molecule break in a number of places and rejoin to form a new ‘blend’ of their mother's and their father's DNA, which is passed on to their own offspring. In contrast, most heterochromatin is inherited without recombining, preserving it in an unaltered form. This is important since recombination in heterochromatin can create genetic abnormalities. Adding small chemical modifications—such as methyl groups—to the histone proteins at the core of the chromatin can change how the DNA is packed. However, the histone modifications that yield different chromatin structures, and the effect of these modifications, are not very well understood. Papazyan et al. have taken advantage of a distinct feature of the protozoan Tetrahymena thermophila: a single-celled organism that divides its chromatin into two different nuclei. The smaller micronuclei contain only heterochromatin, and Papazyan et al. discovered that the histone H3 protein in the micronuclei is modified by methyl groups at a specific site that had not been studied before. Furthermore, this protozoan makes more of these modifications when it reproduces. An enzyme called Ezl3p adds these methyl groups, and without this enzyme T. thermophila reproduces more slowly and has offspring that are less likely to survive and more likely to be infertile. Papazyan et al. provide evidence that these characteristics arise because the cells without the histone modification are unable to prevent DNA breaks from occurring in heterochromatin during recombination. The same histone modification also occurs when the microscopic worm Caenorhabditis elegans reproduces, suggesting that this method of DNA protection has been conserved throughout evolution. Papazyan et al. propose that the histone modification may prevent another enzyme that induces DNA breaks from accessing the heterochromatin in reproductive cells; but more work is required to support this hypothesis. These findings reveal the importance of a new histone modification during reproduction, and could provide new directions for infertility research. DOI: http://dx.doi.org/10.7554/eLife.02996.002

In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.

Heterochromatin and its associated histone modifications are important for repressing transcription and maintaining chromosomal integrity during meiosis and mitosis. The complex repertoire of histone modifications that decorate heterochromatin has yet to be fully characterized, in part because most eukaryotic cells have a single nucleus where distinct chromatin types are intermingled on contiguous stretches of chromosomes. To obtain highly-purified heterochromatin, we turned to the model organism Tetrahymena thermophila, which has a biochemically separable heterochromatic micronucleus. We characterized combinatorial histone modifications on heterochromatic H3 from Tetrahymena and identified species of H3 dually-modified by both H3K23me3 and H3K27me3 as a previously unreported binary ‘mark’ specific for heterochromatin. Furthermore, H3K23me3 levels dramatically increased during meiosis in Tetrahymena micronuclei, C. elegans, and mice, suggesting this histone ‘mark’ plays a conserved role in germline development. Lastly, disrupting the H3K23 methyltransferase in Tetrahymena caused a lag in meiotic progression. Together, our data suggests H3K23me3 is a conserved heterochromatic histone PTM strongly associated with meiosis, and misregulation of this modification may be linked to problems with reproductive fitness and development.