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.

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.

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 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.

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 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.

Osteoporosis- and obesity-risk interrelationships: an epigenetic analysis of GWAS-derived SNPs at the developmental gene TBX15.

A major challenge in translating findings from genome-wide association studies (GWAS) to biological mechanisms is pinpointing functional variants because only a very small percentage of variants associated with a given trait actually impact the trait. We used an extensive epigenetics, transcriptomics, and genetics analysis of the TBX15/WARS2 neighbourhood to prioritize this region's best-candidate causal variants for the genetic risk of osteoporosis (estimated bone density, eBMD) and obesity (waist-hip ratio or waist circumference adjusted for body mass index). TBX15 encodes a transcription factor that is important in bone development and adipose biology. Manual curation of 692 GWAS-derived variants gave eight strong candidates for causal SNPs that modulate TBX15 transcription in subcutaneous adipose tissue (SAT) or osteoblasts, which highly and specifically express this gene. None of these SNPs were prioritized by Bayesian fine-mapping. The eight regulatory causal SNPs were in enhancer or promoter chromatin seen preferentially in SAT or osteoblasts at TBX15 intron-1 or upstream. They overlap strongly predicted, allele-specific transcription factor binding sites. Our analysis suggests that these SNPs act independently of two missense SNPs in TBX15. Remarkably, five of the regulatory SNPs were associated with eBMD and obesity and had the same trait-increasing allele for both. We found that WARS2 obesity-related SNPs can be ascribed to high linkage disequilibrium with TBX15 intron-1 SNPs. Our findings from GWAS index, proxy, and imputed SNPs suggest that a few SNPs, including three in a 0.7-kb cluster, act as causal regulatory variants to fine-tune TBX15 expression and, thereby, affect both obesity and osteoporosis risk.

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.

Data showing atherosclerosis-associated differentially methylated regions are often at enhancers.

Atherosclerosis involves phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs). Data are given in tabular or figure format that illustrate genome-wide DNA methylation alterations in atherosclerotic vs. control aorta (athero DMRs). Data based upon publicly available chromatin state profiles are also shown for normal aorta, monocyte, and skeletal muscle tissue-specific DMRs and for aorta-specific chromatin features (enhancer chromatin, promoter chromatin, repressed chromatin, actively transcribed chromatin). Athero hypomethylated and hypermethylated DMRs as well as epigenetic and transcription profiles are described for the following genes: ACTA2, MYH10, MYH11 (SMC-associated genes); SMAD3 (a signaling gene for SMCs and other cell types); CD79B and SH3BP2 (leukocyte-associated genes); and TBX20 and genes in the HOXA, HOXB, HOXC, and HOXD clusters (T-box and homeobox developmental genes). The data reveal strong correlations between athero hypermethylated DMRs and regions of enhancer chromatin in aorta, which are discussed in the linked research article "Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers" (M. Lacey et al., 2019).

Tissue-specific epigenetics of atherosclerosis-related ANGPT and ANGPTL genes.

AIM: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) genes (especially the five genes embedded in introns of host genes) and their association with atherosclerosis. METHODS: Transcription and epigenomic databases from various normal tissues were examined in the vicinity of ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. RESULTS: We identified tissue-specific enhancer chromatin regions that are likely to regulate transcription of ANGPT/ANGPTL genes and were intragenic, intergenic or host gene-linked. In addition, we found atherosclerosis-linked differentially methylated regions associated with ANGPT2 and with sequences encoding miR-145, a microRNA that targets ANGPT2 mRNA in cancers. CONCLUSION: Our findings implicate enhancers as major contributors to tissue-specific expression of ANGPT/ANGPTL genes, which play critical roles in angiogenesis, atherosclerosis, cancer, and inflammatory and metabolic diseases.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.

BACKGROUND AND AIMS: Atherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. METHODS: We examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. RESULTS: We found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis- and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. CONCLUSIONS: We provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions.

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription.

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression.

Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study-owing to their myogenic DMRs-overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage.

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.

The Aspergillus flavus fluP-associated metabolite promotes sclerotial production.

Aspergillus flavus is able to synthesize a variety of polyketide-derived secondary metabolites including the hepatocarcinogen, aflatoxin B1. The fungus reproduces and disseminates predominantly by production of conidia. It also produces hardened mycelial aggregates called sclerotia that are used to cope with unfavourable growth environments. In the present study, we examined the role of A. flavus fluP, the backbone polyketide synthase gene of secondary metabolite gene cluster 41, on fungal development. The A. flavus CA14 fluP deletion mutant (AfΔfluP) grew and accumulated aflatoxin normally but produced a lower amount of sclerotia than the parental strain. This was also true for the Aspergillus parasiticus BN9 fluP deletion mutant (ApΔfluP). The A. flavus fluP gene was positively regulated by developmental regulators of VeA and VelB but not by the global regulator of secondary metabolism, LaeA. Overexpression of fluP in AfΔfluP (OEfluP) elevated its ability to produce sclerotia compared to that of the parental strain. Coculture of OEfluP with CA14, AfΔfluP, ApΔfluP, or an A. flavus pptA deletion mutant incapable of producing functional polyketide synthases also allowed increased sclerotial production of the respective strains at edges where colonies made contact. Acetone extracts of OEfluP but not of AfΔfluP exhibited the same effect in promoting sclerotial production of AfΔfluP. These results suggest that FluP polyketide synthase is involved in the synthesis of a diffusible metabolite that could serve as a signal molecule to regulate sclerotiogenesis.

Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus.

The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic de-replication of the accurate mass data, five other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple-stage MS data.

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.

Regulation of the aflatoxin-like toxin dothistromin by AflJ.

Biosynthesis by Aspergillus parasiticus of aflatoxin, one of the most potent known naturally occurring carcinogens, requires the activity of two regulatory proteins, AflR and AflJ, which are encoded by divergently transcribed genes within the aflatoxin gene cluster. Although the Zn2Cys6 transcription factor, AflR, has been well-studied, the role of AflJ as a transcription regulatory factor is not well understood. An AflJ-like gene (DsAflJ) is also present in the genome of the pine needle pathogen Dothistroma septosporum and is similarly divergently transcribed from an AflR orthologue (DsAflR). These genes are involved in biosynthesis of dothistromin, a toxic virulence factor related to aflatoxin. DsAflJ mutants produced low levels of dothistromin (<25-fold less than wild-type); this was in contrast to earlier work with A. parasiticus AflJ mutants in which aflatoxin production was more severely impaired. As expected, complementation of D. septosporum mutants with an intact copy of the DsAflJ gene regained production of wild-type levels of dothistromin, although levels were not further increased by over-expression in multi-copy strains. However, heterologous AflJ genes from Aspergillus spp. were unable to complement DsAflJ mutants, suggesting that the proteins function differently in these species.

Aflatoxin biosynthesis is a novel source of reactive oxygen species--a potential redox signal to initiate resistance to oxidative stress?

Aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus involves a minimum of 21 enzymes, encoded by genes located in a 70 kb gene cluster. For aflatoxin biosynthesis to be completed, the required enzymes must be transported to specialized early and late endosomes called aflatoxisomes. Of particular significance, seven aflatoxin biosynthetic enzymes are P450/monooxygenases which catalyze reactions that can produce reactive oxygen species (ROS) as byproducts. Thus, oxidative reactions in the aflatoxin biosynthetic pathway could potentially be an additional source of intracellular ROS. The present work explores the hypothesis that the aflatoxin biosynthetic pathway generates ROS (designated as "secondary" ROS) in endosomes and that secondary ROS possess a signaling function. We used specific dyes that stain ROS in live cells and demonstrated that intracellular ROS levels correlate with the levels of aflatoxin synthesized. Moreover, feeding protoplasts with precursors of aflatoxin resulted in the increase in ROS generation. These data support the hypothesis. Our findings also suggest that secondary ROS may fulfill, at least in part, an important mechanistic role in increased tolerance to oxidative stress in germinating spores (seven-hour germlings) and in regulation of fungal development.