Epigenomic and Transcriptomic Prioritization of Candidate Obesity-Risk Regulatory GWAS SNPs.

Concern about rising rates of obesity has prompted searches for obesity-related single nucleotide polymorphisms (SNPs) in genome-wide association studies (GWAS). Identifying plausible regulatory SNPs is very difficult partially because of linkage disequilibrium. We used an unusual epigenomic and transcriptomic analysis of obesity GWAS-derived SNPs in adipose versus heterologous tissues. From 50 GWAS and 121,064 expanded SNPs, we prioritized 47 potential causal regulatory SNPs (Tier-1 SNPs) for 14 gene loci. A detailed examination of seven loci revealed that four (CABLES1, PC, PEMT, and FAM13A) had Tier-1 SNPs positioned so that they could regulate use of alternative transcription start sites, resulting in different polypeptides being generated or different amounts of an intronic microRNA gene being expressed. HOXA11 and long noncoding RNA gene RP11-392O17.1 had Tier-1 SNPs in their 3' or promoter region, respectively, and strong preferences for expression in subcutaneous versus visceral adipose tissue. ZBED3-AS1 had two intragenic Tier-1 SNPs, each of which could contribute to mediating obesity risk through modulating long-distance chromatin interactions. Our approach not only revealed especially credible novel regulatory SNPs, but also helped evaluate previously highlighted obesity GWAS SNPs that were candidates for transcription regulation.

Epigenetics of Muscle- and Brain-Specific Expression of KLHL Family Genes.

KLHL and the related KBTBD genes encode components of the Cullin-E3 ubiquitin ligase complex and typically target tissue-specific proteins for degradation, thereby affecting differentiation, homeostasis, metabolism, cell signaling, and the oxidative stress response. Despite their importance in cell function and disease (especially, KLHL40, KLHL41, KBTBD13, KEAP1, and ENC1), previous studies of epigenetic factors that affect transcription were predominantly limited to promoter DNA methylation. Using diverse tissue and cell culture whole-genome profiles, we examined 17 KLHL or KBTBD genes preferentially expressed in skeletal muscle or brain to identify tissue-specific enhancer and promoter chromatin, open chromatin (DNaseI hypersensitivity), and DNA hypomethylation. Sixteen of the 17 genes displayed muscle- or brain-specific enhancer chromatin in their gene bodies, and most exhibited specific intergenic enhancer chromatin as well. Seven genes were embedded in super-enhancers (particularly strong, tissue-specific clusters of enhancers). The enhancer chromatin regions typically displayed foci of DNA hypomethylation at peaks of open chromatin. In addition, we found evidence for an intragenic enhancer in one gene upregulating expression of its neighboring gene, specifically for KLHL40/HHATL and KLHL38/FBXO32 gene pairs. Many KLHL/KBTBD genes had tissue-specific promoter chromatin at their 5' ends, but surprisingly, two (KBTBD11 and KLHL31) had constitutively unmethylated promoter chromatin in their 3' exons that overlaps a retrotransposed KLHL gene. Our findings demonstrate the importance of expanding epigenetic analyses beyond the 5' ends of genes in studies of normal and abnormal gene regulation.

DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery.

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

Epigenetics of Genes Preferentially Expressed in Dissimilar Cell Populations: Myoblasts and Cerebellum.

While studying myoblast methylomes and transcriptomes, we found that CDH15 had a remarkable preference for expression in both myoblasts and cerebellum. To understand how widespread such a relationship was and its epigenetic and biological correlates, we systematically looked for genes with similar transcription profiles and analyzed their DNA methylation and chromatin state and accessibility profiles in many different cell populations. Twenty genes were expressed preferentially in myoblasts and cerebellum (Myob/Cbl genes). Some shared DNA hypo- or hypermethylated regions in myoblasts and cerebellum. Particularly striking was ZNF556, whose promoter is hypomethylated in expressing cells but highly methylated in the many cell populations that do not express the gene. In reporter gene assays, we demonstrated that its promoter's activity is methylation sensitive. The atypical epigenetics of ZNF556 may have originated from its promoter's hypomethylation and selective activation in sperm progenitors and oocytes. Five of the Myob/Cbl genes (KCNJ12, ST8SIA5, ZIC1, VAX2, and EN2) have much higher RNA levels in cerebellum than in myoblasts and displayed myoblast-specific hypermethylation upstream and/or downstream of their promoters that may downmodulate expression. Differential DNA methylation was associated with alternative promoter usage for Myob/Cbl genes MCF2L, DOK7, CNPY1, and ANK1. Myob/Cbl genes PAX3, LBX1, ZNF556, ZIC1, EN2, and VAX2 encode sequence-specific transcription factors, which likely help drive the myoblast and cerebellum specificity of other Myob/Cbl genes. This study extends our understanding of epigenetic/transcription associations related to differentiation and may help elucidate relationships between epigenetic signatures and muscular dystrophies or cerebellar-linked neuropathologies.

Aspergillus flavus VelB acts distinctly from VeA in conidiation and may coordinate with FluG to modulate sclerotial production.

The proteins VeA, VelB and LaeA of Aspergillus nidulans form a heterotrimeric complex (the velvet complex) in the dark to coordinate sexual development and production of some secondary metabolites. VeA and VelB of A. nidulans and Aspergillus fumigatus also are repressors of conidiation, but VeA of Aspergillus flavus in studied strains acts positively on conidiation. In the present study, we show via yeast-two hybrid assays that interactions among A. flavus VeA, VelB, and LaeA are conserved as in the A. nidulans velvet complex. We found that FluG, which is required for conidiophore formation in A. nidulans but whose deletion in A. flavus delays onset of conidiation, was probably an interacting partner of VelB. Deletion of velB in A. flavus CA14 severely impaired conidiation in the dark although to a lesser extent than deletion of veA. In both mutants fluG deletion resulted in further decreased conidiation even in the light. Deletion of fluG in the ΔlaeA strain, however, did not affect conidiation. All mutant types were unable to produce aflatoxin and sclerotia. Cross-complementation of the ΔvelB strain with gpdA::veA restored conidiation but not aflatoxin production although aflR, the aflatoxin pathway regulatory gene, was expressed at a normal level. Cross-complementation of the ΔveA strain with gpdA::velB failed to restore conidiation and aflatoxin production. The ΔvelB strain complemented with or a wild type transformed by gpdA::velB had elevated sclerotial production as the ΔfluG strain. Concerted interactions of A. flavus VeA and VelB with LaeA are critical for conidiation and aflatoxin biosynthesis. VelB may have a dual role and likely coordinates with FluG to modulate sclerotial production.

Dothistromin genes at multiple separate loci are regulated by AflR.

In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (ΔDsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ΔAflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ΔDsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ΔDsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.

Fragmentation of an aflatoxin-like gene cluster in a forest pathogen.

Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D. septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D. septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.

Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus.

Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5₋12-Cys-X2-Cys-X6₋9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overview of this family of genes. Annotation of this gene family in most fungal genomes is still far from perfect and refined bioinformatic algorithms are urgently needed. Aspergillus flavus is a saprophytic soil fungus that can produce the carcinogenic aflatoxin. It is the second leading causative agent of invasive aspergillosis. The 37-Mb genome of A. flavus is predicted to encode 12,000 proteins. Two and a half percent of the total proteins are estimated to contain the C6 domain, more than twofold greater than those estimated for yeast, which is about 1 %. The variability in the spacing between cysteines, C3-C4 and C5-C6, in the zinc cluster enables classification of the domains into distinct subgroups, which are also well conserved in Aspergillus nidulans. Sixty-six percent (202/306) of the A. flavus C6 proteins contain a specific transcription factor domain, and 7 % contain a domain of unknown function, DUF3468. Two A. nidulans C6 proteins containing the DUF3468 are involved in asexual conidiation and another two in sexual differentiation. In the anamorphic A. flavus, a homolog of the latter lacks the C6 domain. A. flavus being heterothallic and reproducing mainly through conidiation appears to have lost some components involved in homothallic sexual development. Of the 55 predicted gene clusters thought to be involved in production of secondary metabolites, only about half have a C6-encoding gene in or near the gene clusters. The features revealed by the A. flavus C6 proteins likely are common for other ascomycete fungi.

NsdC and NsdD affect Aspergillus flavus morphogenesis and aflatoxin production.

The transcription factors NsdC and NsdD are required for sexual development in Aspergillus nidulans. We now show these proteins also play a role in asexual development in the agriculturally important aflatoxin (AF)-producing fungus Aspergillus flavus. We found that both NsdC and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Conidiophores in nsdC and nsdD deletion mutants had shortened stipes and altered conidial heads compared to those of wild-type A. flavus. Our results suggest that NsdC and NsdD regulate transcription of genes required for early processes in conidiophore development preceding conidium formation. As the cultures aged, the ΔnsdC and ΔnsdD mutants produced a dark pigment that was not observed in the wild type. Gene expression data showed that although AflR is expressed at normal levels, a number of aflatoxin biosynthesis genes are expressed at reduced levels in both nsd mutants. Expression of aflD, aflM, and aflP was greatly reduced in nsdC mutants, and neither aflatoxin nor the proteins for these genes could be detected. Our results support previous studies showing that there is a strong association between conidiophore and sclerotium development and aflatoxin production in A. flavus.

Deletion of the Aspergillus flavus orthologue of A. nidulans fluG reduces conidiation and promotes production of sclerotia but does not abolish aflatoxin biosynthesis.

The fluG gene is a member of a family of genes required for conidiation and sterigmatocystin production in Aspergillus nidulans. We examined the role of the Aspergillus flavus fluG orthologue in asexual development and aflatoxin biosynthesis. Deletion of fluG in A. flavus yielded strains with an approximately 3-fold reduction in conidiation but a 30-fold increase in sclerotial formation when grown on potato dextrose agar in the dark. The concurrent developmental changes suggest that A. flavus FluG exerts opposite effects on a mutual signaling pathway for both processes. The altered conidial development was in part attributable to delayed expression of brlA, a gene controlling conidiophore formation. Unlike the loss of sterigmatocystin production by A. nidulans fluG deletion strains, aflatoxin biosynthesis was not affected by the fluG deletion in A. flavus. In A. nidulans, FluG was recently found to be involved in the formation of dehydroaustinol, a component of a diffusible signal of conidiation. Coculturing experiments did not show a similar diffusible meroterpenoid secondary metabolite produced by A. flavus. These results suggest that the function of fluG and the signaling pathways related to conidiation are different in the two related aspergilli.

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog.

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.

Promoter-Adjacent DNA Hypermethylation Can Downmodulate Gene Expression: TBX15 in the Muscle Lineage.

TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection. These cloned regions exhibited strong promoter activity and, when placed upstream of a weak promoter, strong enhancer activity specifically in myoblast host cells. In vitro CpG methylation targeted to the DMR sequences in the plasmids resulted in 86−100% loss of promoter or enhancer activity, depending on the insert sequence. These results as well as chromatin epigenetic and transcription profiles for this gene in various cell types support the hypothesis that DNA hypermethylation immediately upstream and downstream of the unmethylated promoter region suppresses enhancer/extended promoter activity, thereby downmodulating, but not silencing, expression in myoblasts and certain kinds of skeletal muscle. This promoter-border hypermethylation was not found in cell types with a silent TBX15 gene, and these cells, instead, exhibit repressive chromatin in and around the promoter. TBX18, TBX2, TBX3 and TBX1 display TBX15-like hypermethylated DMRs at their promoter borders and preferential expression in myoblasts. Therefore, promoter-adjacent DNA hypermethylation for downmodulating transcription to prevent overexpression may be used more frequently for transcription regulation than currently appreciated.

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus.

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).

Epigenetics of Mitochondria-Associated Genes in Striated Muscle.

Striated muscle has especially large energy demands. We identified 97 genes preferentially expressed in skeletal muscle and heart, but not in aorta, and found significant enrichment for mitochondrial associations among them. We compared the epigenomic and transcriptomic profiles of the 27 genes associated with striated muscle and mitochondria. Many showed strong correlations between their tissue-specific transcription levels, and their tissue-specific promoter, enhancer, or open chromatin as well as their DNA hypomethylation. Their striated muscle-specific enhancer chromatin was inside, upstream, or downstream of the gene, throughout much of the gene as a super-enhancer (CKMT2, SLC25A4, and ACO2), or even overlapping a neighboring gene (COX6A2, COX7A1, and COQ10A). Surprisingly, the 3' end of the 1.38 Mb PRKN (PARK2) gene (involved in mitophagy and linked to juvenile Parkinson's disease) displayed skeletal muscle/myoblast-specific enhancer chromatin, a myoblast-specific antisense RNA, as well as brain-specific enhancer chromatin. We also found novel tissue-specific RNAs in brain and embryonic stem cells within PPARGC1A (PGC-1α), which encodes a master transcriptional coregulator for mitochondrial formation and metabolism. The tissue specificity of this gene's four alternative promoters, including a muscle-associated promoter, correlated with nearby enhancer chromatin and open chromatin. Our in-depth epigenetic examination of these genes revealed previously undescribed tissue-specific enhancer chromatin, intragenic promoters, regions of DNA hypomethylation, and intragenic noncoding RNAs that give new insights into transcription control for this medically important set of genes.

Epigenetics and expression of key genes associated with cardiac fibrosis: NLRP3, MMP2, MMP9, CCN2/CTGF and AGT.

Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix drive cardiac fibrosis and require changes in gene expression. Materials and methods: Using bioinformatics, both tissue-specific expression profiles and epigenomic profiles of some genes critical for cardiac fibrosis were examined, namely, NLRP3, MMP2, MMP9, CCN2/CTGF, AGT (encodes angiotensin II precursors) and hsa-mir-223 (post-transcriptionally regulates NLRP3). Results: In monocytes, neutrophils, fibroblasts, venous cells, liver and brain, enhancers or super-enhancers were found that correlate with high expression of these genes. One enhancer extended into a silent gene neighbor. These enhancers harbored tissue-specific foci of DNA hypomethylation, open chromatin and transcription factor binding. Conclusions: This study identified previously undescribed enhancers containing hypomethylated transcription factor binding subregions that are predicted to regulate expression of these cardiac fibrosis-inducing genes.

HypC, the anthrone oxidase involved in aflatoxin biosynthesis.

On the basis of gene disruption and enzyme activity, hypC, an open reading frame in the region between the pksA (aflC) and nor-1 (aflD) genes in the aflatoxin biosynthesis gene cluster, encodes a 17-kDa oxidase that converts norsolorinic acid anthrone to norsolorinic acid.

Epigenetics of Skeletal Muscle-Associated Genes in the ASB, LRRC, TMEM, and OSBPL Gene Families.

Much remains to be discovered about the intersection of tissue-specific transcription control and the epigenetics of skeletal muscle (SkM), a very complex and dynamic organ. From four gene families, Leucine-Rich Repeat Containing (LRRC), Oxysterol Binding Protein Like (OSBPL), Ankyrin Repeat and Socs Box (ASB), and Transmembrane Protein (TMEM), we chose 21 genes that are preferentially expressed in human SkM relative to 52 other tissue types and analyzed relationships between their tissue-specific epigenetics and expression. We also compared their genetics, proteomics, and descriptions in the literature. For this study, we identified genes with little or no previous descriptions of SkM functionality (ASB4, ASB8, ASB10, ASB12, ASB16, LRRC14B, LRRC20, LRRC30, TMEM52, TMEM233, OSBPL6/ORP6, and OSBPL11/ORP11) and included genes whose SkM functions had been previously addressed (ASB2, ASB5, ASB11, ASB15, LRRC2, LRRC38, LRRC39, TMEM38A/TRIC-A, and TMEM38B/TRIC-B). Some of these genes have associations with SkM or heart disease, cancer, bone disease, or other diseases. Among the transcription-related SkM epigenetic features that we identified were: super-enhancers, promoter DNA hypomethylation, lengthening of constitutive low-methylated promoter regions, and SkM-related enhancers for one gene embedded in a neighboring gene (e.g., ASB8-PFKM, LRRC39-DBT, and LRRC14B-PLEKHG4B gene-pairs). In addition, highly or lowly co-expressed long non-coding RNA (lncRNA) genes probably regulate several of these genes. Our findings give insights into tissue-specific epigenetic patterns and functionality of related genes in a gene family and can elucidate normal and disease-related regulation of gene expression in SkM.