Disruption of H3K36 methylation provokes cellular plasticity to drive aberrant glandular formation and squamous carcinogenesis.

Chromatin organization is essential for maintaining cell-fate trajectories and developmental programs. Here, we find that disruption of H3K36 methylation dramatically impairs normal epithelial differentiation and development, which promotes increased cellular plasticity and enrichment of alternative cell fates. Specifically, we observe a striking increase in the aberrant generation of excessive epithelial glandular tissues, including hypertrophic salivary, sebaceous, and meibomian glands, as well as enhanced squamous tumorigenesis. These phenotypic and gene expression manifestations are associated with loss of H3K36me2 and rewiring of repressive H3K27me3, changes we also observe in human patients with glandular hyperplasia. Collectively, these results have identified a critical role for H3K36 methylation in both in vivo epithelial cell-fate decisions and the prevention of squamous carcinogenesis and suggest that H3K36 methylation modulation may offer new avenues for the treatment of numerous common disorders driven by altered glandular function, which collectively affect large segments of the human population.

Splicing quality control mediated by DHX15 and its G-patch activator SUGP1.

Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identify a widespread splicing QC function for DHX15 in human cells, consistent with recent in vitro studies. We find that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identify SUGP1 as a G-patch factor that activates DHX15's splicing QC function. This interaction is dependent on both DHX15's ATPase activity and on SUGP1's U2AF ligand motif (ULM) domain. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.

Alternative splicing of HDAC7 regulates its interaction with 14-3-3 proteins to alter histone marks and target gene expression.

Chromatin regulation and alternative splicing are both critical mechanisms guiding gene expression. Studies have demonstrated that histone modifications can influence alternative splicing decisions, but less is known about how alternative splicing may impact chromatin. Here, we demonstrate that several genes encoding histone-modifying enzymes are alternatively spliced downstream of T cell signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression and differentiation in T cells. Using CRISPR-Cas9 gene editing and cDNA expression, we show that differential inclusion of HDAC7 exon 9 controls the interaction of HDAC7 with protein chaperones, resulting in changes to histone modifications and gene expression. Notably, the long isoform, which is induced by the RNA-binding protein CELF2, promotes expression of several critical T cell surface proteins including CD3, CD28, and CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on histone modification and gene expression that contributes to T cell development.

Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease.

The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Here, we report a novel RNA binding function for Tip60 in addition to its HAT function. We show that Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, we investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. Our results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) has recently emerged as a hotbed for RNA alternative splicing (AS) defects that alter protein function in the brain yet causes remain unclear. Although recent findings suggest convergence of epigenetics with co-transcriptional AS, whether epigenetic dysregulation in AD pathology underlies AS defects remains unknown. Here, we identify a novel RNA interaction and splicing regulatory function for Tip60 histone acetyltransferase (HAT) that is disrupted in Drosophila brains modeling AD pathology and in human AD hippocampus. Importantly, mammalian orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brain. We propose that Tip60-mediated AS modulation is a conserved critical posttranscriptional step that may underlie AS defects now characterized as hallmarks of AD.

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice.

Cocaine epigenetically regulates gene expression via changes in histone post-translational modifications (HPTMs). We previously found that the immediate early gene Nr4a1 is epigenetically activated by cocaine in mouse brain reward regions. However, few studies have examined multiple HPTMs at a single gene. Bivalent gene promoters are simultaneously enriched in both activating (H3K4me3 (K4)) and repressive (H3K27me3 (K27)) HPTMs. As such, bivalent genes are lowly expressed but poised for activity-dependent gene regulation. In this study, we identified K4&K27 bivalency at Nr4a1 following investigator-administered cocaine in male and female mice. We applied sequential chromatin immunoprecipitation and qPCR to define Nr4a1 bivalency and expression in striatum (STR), prefrontal cortex (PFC), and hippocampus (HPC). We used Pearson's correlation to quantify relationships within each brain region across treatment conditions for each sex. In female STR, cocaine increased Nr4a1 mRNA while maintaining Nr4a1 K4&K27 bivalency. In male STR, cocaine enriched repressive H3K27me3 and K4&K27 bivalency at Nr4a1 and maintained Nr4a1 mRNA. Furthermore, cocaine epigenetically regulated a putative NR4A1 target, Cartpt, in male PFC. This study defined the epigenetic regulation of Nr4a1 in reward brain regions in male and female mice following cocaine, and, thus, shed light on the biological relevance of sex to cocaine use disorder.

Comparative phylotranscriptomics reveals putative sex differentiating genes across eight diverse bivalve species.

Mollusks, especially bivalves, exhibit a great diversity of sex determining mechanisms, including both genetic and environmental sex determination. Some bivalve species can be gonochoristic (separate sexes), while others are hermaphroditic (sequential or simultaneous). Several models have been proposed for specific bivalve species, utilizing information gained from gene expression data, as well as limited RAD-seq data (e.g., from Crassostrea gigas). However, these mechanisms are not as well studied as those in model organisms (e.g., Mus musculus, Drosophila melanogaster, Caenorhabditis elegans) and many genes involved in sex differentiation are not well characterized. We used phylotranscriptomics to better understand which possible sex differentiating genes are in bivalves and how these genes relate to similar genes in diverse phyla. We collected RNAseq data from eight phylogenetically diverse bivalve species: Argopecten irradians, Ensis directus, Geukensia demissa, Macoma tenta, Mercenaria mercenaria, Mya arenaria, Mytilus edulis, and Solemya velum. Using these data, we assembled representative transcriptomes for each species. We then searched for candidate sex differentiating genes using BLAST and confirmed the identity of nine genes using phylogenetics analyses from nine phyla. To increase the confidence of identification, we included ten bivalve genomes in our analyses. From the analysis of doublesex and mab-3 related transcription factor (DMRT) genes, we confirmed the identify of a Mollusk-specific sex determining DMRT gene: DMRT1L. Based on gene expression data from M. edulis and previous research, DMRT1L and FoxL2 are key genes for male and female development, respectively.

Developmental Exposure to PCB153 (2,2',4,4',5,5'-Hexachlorobiphenyl) Alters Circadian Rhythms and the Expression of Clock and Metabolic Genes.

Polychlorinated biphenyls (PCBs) are highly persistent and ubiquitously distributed environmental pollutants. Based on their chemical structure, PCBs are classified into non-ortho-substituted and ortho-substituted congeners. Non-ortho-substituted PCBs are structurally similar to dioxin and their toxic effects and mode of action are well-established. In contrast, very little is known about the effects of ortho-substituted PCBs, particularly, during early development. The objective of this study is to investigate the effects of exposure to an environmentally prominent ortho-substituted PCB (2,2',4,4',5,5'-hexachlorobiphenyl; PCB153) on zebrafish embryos. We exposed zebrafish embryos to 3 different concentrations of PCB153 starting from 4 to 120 hours post-fertilization (hpf). We quantified gross morphological changes, behavioral phenotypes, gene expression changes, and circadian behavior in the larvae. There were no developmental defects during the exposure period, but starting at 7 dpf, we observed spinal deformity in the 10 µM PCB153 treated group. A total of 633, 2227, and 3378 differentially expressed genes were observed in 0.1 µM (0.036 µg/ml), 1 µM (0.36 µg/ml), and 10 µM (3.6 µg/ml) PCB153-treated embryos, respectively. Of these, 301 genes were common to all treatment groups. KEGG pathway analysis revealed enrichment of genes related to circadian rhythm, FoxO signaling, and insulin resistance pathways. Behavioral analysis revealed that PCB153 exposure significantly alters circadian behavior. Disruption of circadian rhythms has been associated with the development of metabolic and neurological diseases. Thus, understanding the mechanisms of action of environmental chemicals in disrupting metabolism and other physiological processes is essential.

Exon-Mediated Activation of Transcription Starts.

The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals.

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis.

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed Mytilus edulis to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 µg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an M. edulis microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field.

Role of DNA methylation in altered gene expression patterns in adult zebrafish (Danio rerio) exposed to 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB 126).

There is growing evidence that environmental toxicants can affect various physiological processes by altering DNA methylation patterns. However, very little is known about the impact of toxicant-induced DNA methylation changes on gene expression patterns. The objective of this study was to determine the genome-wide changes in DNA methylation concomitant with altered gene expression patterns in response to 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB126) exposure. We used PCB126 as a model environmental chemical because the mechanism of action is well-characterized, involving activation of aryl hydrocarbon receptor, a ligand-activated transcription factor. Adult zebrafish were exposed to 10 nM PCB126 for 24 h (water-borne exposure) and brain and liver tissues were sampled at 7 days post-exposure in order to capture both primary and secondary changes in DNA methylation and gene expression. We used enhanced Reduced Representation Bisulfite Sequencing and RNAseq to quantify DNA methylation and gene expression, respectively. Enhanced reduced representation bisulfite sequencing analysis revealed 573 and 481 differentially methylated regions in the liver and brain, respectively. Most of the differentially methylated regions are located more than 10 kilobases upstream of transcriptional start sites of the nearest neighboring genes. Gene Ontology analysis of these genes showed that they belong to diverse physiological pathways including development, metabolic processes and regeneration. RNAseq results revealed differential expression of genes related to xenobiotic metabolism, oxidative stress and energy metabolism in response to polychlorinated biphenyl exposure. There was very little correlation between differentially methylated regions and differentially expressed genes suggesting that the relationship between methylation and gene expression is dynamic and complex, involving multiple layers of regulation.