Recognition of Histone Crotonylation by Taf14 Links Metabolic State to Gene Expression.

Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding.

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60 bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits.

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response.

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.

Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction.

AIMS: Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeller gene Ino80 results in defective coronary vessel development that leads to ventricular non-compaction in embryonic mouse hearts. We aimed to identify candidate angiocrines expressed by endocardial and endothelial cells (ECs) in wildtype and LVNC conditions in Tie2Cre;Ino80fl/fltransgenic embryonic mouse hearts, and test the effect of these candidates on cardiomyocyte proliferation and maturation. METHODS AND RESULTS: We used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behaviour. We identified Col15a1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells up-regulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation. CONCLUSIONS: These findings support a model where coronary endothelial cells normally promote myocardial compaction through secreted factors, but that endocardial and endothelial cells can secrete factors that contribute to non-compaction under pathological conditions.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes.

Chromatin-modifying factors have essential roles in DNA processing pathways that dictate cellular functions. The ability of chromatin modifiers, including the INO80 and SWR1 chromatin-remodelling complexes, to regulate transcriptional processes is well established. However, recent studies reveal that the INO80 and SWR1 complexes have crucial functions in many other essential processes, including DNA repair, checkpoint regulation, DNA replication, telomere maintenance and chromosome segregation. During these diverse nuclear processes, the INO80 and SWR1 complexes function cooperatively with their histone substrates, gamma-H2AX and H2AZ. This research reveals that INO80 and SWR1 ATP-dependent chromatin remodelling is an integral component of pathways that maintain genomic integrity.

The INO80 chromatin remodeler sustains metabolic stability by promoting TOR signaling and regulating histone acetylation.

Chromatin remodeling complexes are essential for gene expression programs that coordinate cell function with metabolic status. However, how these remodelers are integrated in metabolic stability pathways is not well known. Here, we report an expansive genetic screen with chromatin remodelers and metabolic regulators in Saccharomyces cerevisiae. We found that, unlike the SWR1 remodeler, the INO80 chromatin remodeling complex is composed of multiple distinct functional subunit modules. We identified a strikingly divergent genetic signature for the Ies6 subunit module that links the INO80 complex to metabolic homeostasis. In particular, mitochondrial maintenance is disrupted in ies6 mutants. INO80 is also needed to communicate TORC1-mediated signaling to chromatin, as ino80 mutants exhibit defective transcriptional profiles and altered histone acetylation of TORC1-responsive genes. Furthermore, comparative analysis reveals subunits of INO80 and mTORC1 have high co-occurrence of alterations in human cancers. Collectively, these results demonstrate that the INO80 complex is a central component of metabolic homeostasis that influences histone acetylation and may contribute to disease when disrupted.

Glyphosate exposure in pregnancy and shortened gestational length: a prospective Indiana birth cohort study.

BACKGROUND: Glyphosate (GLY) is the most heavily used herbicide worldwide but the extent of exposure in human pregnancy remains unknown. Its residues are found in the environment, major crops, and food items that humans, including pregnant women, consume daily. Since GLY exposure in pregnancy may also increase fetal exposure risk, we designed a birth-cohort study to determine exposure frequency, potential exposure pathways, and associations with fetal growth indicators and pregnancy length. METHOD: Urine and residential drinking water samples were obtained from 71 women with singleton pregnancies living in Central Indiana while they received routine prenatal care. GLY measurements were performed using liquid chromatography-tandem mass spectrometry. Demographic and survey information relating to food and water consumption, stress, and residence were obtained by questionnaire. Maternal risk factors and neonatal outcomes were abstracted from medical records. Correlation analyses were used to assess relationships of urine GLY levels with fetal growth indicators and gestational length. RESULTS: The mean age of participants was 29 years, and the majority were Caucasian. Ninety three percent of the pregnant women had GLY levels above the limit of detection (0.1 ng/mL). Mean urinary GLY was 3.40 ng/mL (range 0.5-7.20 ng/mL). Higher GLY levels were found in women who lived in rural areas (p = 0.02), and in those who consumed > 24 oz. of caffeinated beverages per day (p = 0.004). None of the drinking water samples had detectable GLY levels. We observed no correlations with fetal growth indicators such as birth weight percentile and head circumference. However, higher GLY urine levels were significantly correlated with shortened gestational lengths (r = - 0.28, p = 0.02). CONCLUSIONS: This is the first study of GLY exposure in US pregnant women using urine specimens as a direct measure of exposure. We found that > 90% of pregnant women had detectable GLY levels and that these levels correlated significantly with shortened pregnancy lengths. Although our study cohort was small and regional and had limited racial/ethnic diversity, it provides direct evidence of maternal GLY exposure and a significant correlation with shortened pregnancy. Further investigations in a more geographically and racially diverse cohort would be necessary before these findings could be generalized.

Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities.

ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement.

HIV testing uptake and acceptability in an inner city polyclinic.

Up to 33% of HIV-infected adults in the UK remain undiagnosed and efforts to increase HIV testing are underway. HIV testing was conducted amongst individuals presenting to a polyclinic at a central London hospital using a point of care test. Demographic and HIV risk data was collected along with a patient feedback questionnaire exploring acceptability of the HIV testing experience. Seventy-one out of 93 (76%) individuals accepted HIV testing. Of those accepting HIV testing, 53/71 (75%) had never previously tested for HIV despite, 45/53 (85%) of these being registered with a GP. Twenty-seven out of 71 (38%) of individuals testing had at least one risk factor associated with HIV acquisition, and of these 17/27 (63%) had never previously tested for HIV infection. There were no new HIV positive diagnoses during the period of testing. Respondents indicated a high level of satisfaction with the service and more than 85% found the service to be helpful, educational and convenient. This small proof of concept pilot showed uptake of HIV testing in this setting to be high and acceptable to patients.

Cancer cell metabolism connects epigenetic modifications to transcriptional regulation.

Adaptation of cellular function with the nutrient environment is essential for survival. Failure to adapt can lead to cell death and/or disease. Indeed, energy metabolism alterations are a major contributing factor for many pathologies, including cancer, cardiovascular disease, and diabetes. In particular, a primary characteristic of cancer cells is altered metabolism that promotes survival and proliferation even in the presence of limited nutrients. Interestingly, recent studies demonstrate that metabolic pathways produce intermediary metabolites that directly influence epigenetic modifications in the genome. Emerging evidence demonstrates that metabolic processes in cancer cells fuel malignant growth, in part, through epigenetic regulation of gene expression programs important for proliferation and adaptive survival. In this review, recent progress toward understanding the relationship of cancer cell metabolism, epigenetic modification, and transcriptional regulation will be discussed. Specifically, the need for adaptive cell metabolism and its modulation in cancer cells will be introduced. Current knowledge on the emerging field of metabolite production and epigenetic modification will also be reviewed. Alterations of DNA (de)methylation, histone modifications, such as (de)methylation and (de)acylation, as well as chromatin remodeling, will be discussed in the context of cancer cell metabolism. Finally, how these epigenetic alterations contribute to cancer cell phenotypes will be summarized. Collectively, these studies reveal that both metabolic and epigenetic pathways in cancer cells are closely linked, representing multiple opportunities to therapeutically target the unique features of malignant growth.

Retinoblastoma protein regulates carcinogen susceptibility at heterochromatic cancer driver loci.

Carcinogenic insult, such as UV light exposure, creates DNA lesions that evolve into mutations if left unrepaired. These resulting mutations can contribute to carcinogenesis and drive malignant phenotypes. Susceptibility to carcinogens (i.e., the propensity to form a carcinogen-induced DNA lesion) is regulated by both genetic and epigenetic factors. Importantly, carcinogen susceptibility is a critical contributor to cancer mutagenesis. It is known that mutations can be prevented by tumor suppressor regulation of DNA damage response pathways; however, their roles carcinogen susceptibility have not yet been reported. In this study, we reveal that the retinoblastoma (RB1) tumor suppressor regulates UV susceptibility across broad regions of the genome. In particular, centromere and telomere-proximal regions exhibit significant increases in UV lesion susceptibility when RB1 is deleted. Several cancer-related genes are located within genomic regions of increased susceptibility, including telomerase reverse transcriptase, TERT, thereby accelerating mutagenic potential in cancers with RB1 pathway alterations. These findings reveal novel genome stability mechanisms of a tumor suppressor and uncover new pathways to accumulate mutations during cancer evolution.

Pharmacokinetic and safety profile of raltegravir and ribavirin, when dosed separately and together, in healthy volunteers.

BACKGROUND: Treatment of chronic hepatitis C virus (HCV) infection in HIV-1-co-infected individuals remains challenging due to numerous factors, including drug-drug interactions. The aim of this study was to assess the safety and pharmacokinetic (PK) profile of raltegravir and ribavirin when dosed separately and together. METHODS: Fourteen healthy volunteers [mean (standard deviation) age 35 (10) years, 71% male] entered this phase 1 PK study and received single-dose ribavirin (800 mg) on day 1 (phase 1). Following a washout period, subjects received raltegravir (400 mg twice daily) on days 15-19 (phase 2) and single-dose ribavirin (800 mg) with raltegravir (400 mg) on day 20 (phase 3). Intensive PK sampling was undertaken on days 1, 19 and 20 and differences in geometric mean ratios (GMRs) for PK parameters between study periods were assessed. RESULTS: No statistically significant differences in PK parameters were observed for raltegravir between phases 2 and 3. A statistically significant decrease in maximum plasma concentration (C(max)) and an increase in time to maximum plasma concentration (T(max)) were observed for ribavirin in phase 3 compared with phase 1 [GMR (95% confidence interval) 0.79 (0.62-1.00) and 1.39 (1.08-1.78), respectively], whereas no significant differences in other ribavirin PK parameters were observed between study phases. No clinically significant safety concerns were reported. CONCLUSIONS: The PK profile of ribavirin is altered when administered with raltegravir (reduced C(max) and increased T(max)), with no safety concerns identified. This is unlikely to be of clinical significance or have an impact on the antiviral effects of ribavirin in HIV-1- and HCV-co-infected subjects.

Function of microtubules in intercellular transport of plant virus RNA.

Cell-to-cell progression of tobacco mosaic virus (TMV) infection in plants depends on virus-encoded movement protein (MP). Here we show that a conserved sequence motif in tobamovirus MPs shares similarity with a region in tubulins that is proposed to mediate lateral contacts between microtubule protofilaments. Point mutations in this motif confer temperature sensitivity to microtubule association and viral-RNA intercellular-transport functions of the protein, indicating that MP-interacting microtubules are functionally involved in the transport of vRNA to plasmodesmata. Moreover, we show that MP interacts with microtubule-nucleation sites. Together, our results indicate that MP may mimic tubulin assembly surfaces to propel vRNA transport by a dynamic process that is driven by microtubule polymerization.

Genome-wide profiles of UV lesion susceptibility, repair, and mutagenic potential in melanoma.

Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma.

Regulation of telomere structure and functions by subunits of the INO80 chromatin remodeling complex.

ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex.

Chromatin-remodeling links metabolic signaling to gene expression.

BACKGROUND: ATP-dependent chromatin remodelers are evolutionarily conserved complexes that alter nucleosome positioning to influence many DNA-templated processes, such as replication, repair, and transcription. In particular, chromatin remodeling can dynamically regulate gene expression by altering accessibility of chromatin to transcription factors. SCOPE OF REVIEW: This review provides an overview of the importance of chromatin remodelers in the regulation of metabolic gene expression. Particular emphasis is placed on the INO80 and SWI/SNF (BAF/PBAF) chromatin remodelers in both yeast and mammals. This review details discoveries from the initial identification of chromatin remodelers in Saccharomyces cerevisiae to recent discoveries in the metabolic requirements of developing embryonic tissues in mammals. MAJOR CONCLUSIONS: INO80 and SWI/SNF (BAF/PBAF) chromatin remodelers regulate the expression of energy metabolism pathways in S. cerevisiae and mammals in response to diverse nutrient environments. In particular, the INO80 complex organizes the temporal expression of gene expression in the metabolically synchronized S. cerevisiae system. INO80-mediated chromatin remodeling is also needed to constrain cell division during metabolically favorable conditions. Conversely, the BAF/PBAF remodeler regulates tissue-specific glycolytic metabolism and is disrupted in cancers that are dependent on glycolysis for proliferation. The role of chromatin remodeling in metabolic gene expression is downstream of the metabolic signaling pathways, such as the TOR pathway, a critical regulator of metabolic homeostasis. Furthermore, the INO80 and BAF/PBAF chromatin remodelers have both been shown to regulate heart development, the tissues of which have unique requirements for energy metabolism during development. Collectively, these results demonstrate that chromatin remodelers communicate metabolic status to chromatin and are a central component of homeostasis pathways that optimize cell fitness, organismal development, and prevent disease.

Somatic mutation distributions in cancer genomes vary with three-dimensional chromatin structure.

Somatic mutations in driver genes may ultimately lead to the development of cancer. Understanding how somatic mutations accumulate in cancer genomes and the underlying factors that generate somatic mutations is therefore crucial for developing novel therapeutic strategies. To understand the interplay between spatial genome organization and specific mutational processes, we studied 3,000 tumor-normal-pair whole-genome datasets from 42 different human cancer types. Our analyses reveal that the change in somatic mutational load in cancer genomes is co-localized with topologically-associating-domain boundaries. Domain boundaries constitute a better proxy to track mutational load change than replication timing measurements. We show that different mutational processes lead to distinct somatic mutation distributions where certain processes generate mutations in active domains, and others generate mutations in inactive domains. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation-rate variations observed in human cancers.

The somatic mutation landscape of the human body.

BACKGROUND: Somatic mutations in healthy tissues contribute to aging, neurodegeneration, and cancer initiation, yet they remain largely uncharacterized. RESULTS: To gain a better understanding of the genome-wide distribution and functional impact of somatic mutations, we leverage the genomic information contained in the transcriptome to uniformly call somatic mutations from over 7500 tissue samples, representing 36 distinct tissues. This catalog, containing over 280,000 mutations, reveals a wide diversity of tissue-specific mutation profiles associated with gene expression levels and chromatin states. For example, lung samples with low expression of the mismatch-repair gene MLH1 show a mutation signature of deficient mismatch repair. In addition, we find pervasive negative selection acting on missense and nonsense mutations, except for mutations previously observed in cancer samples, which are under positive selection and are highly enriched in many healthy tissues. CONCLUSIONS: These findings reveal fundamental patterns of tissue-specific somatic evolution and shed light on aging and the earliest stages of tumorigenesis.

Initial Findings From an Acute Hospital Care at Home Waiver Initiative.

This cohort study assesses outcomes of patients treated during the initial 16 months of the Centers for Medicare & Medicaid Services Acute Hospital Care at Home initiative.