Proximal Regulatory Region Research Articles

Mammalian ubiquitous promoter isolated from proximal regulatory region of bovine MSTN gene.

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations

BackgroundRecent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype.ResultsIn this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases.ConclusionsUsing our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Integrative analysis of transcriptomic and epigenomic data reveals distinct patterns for developmental and housekeeping gene regulation

BackgroundRegulation of transcription is central to the emergence of new cell types during development, and it often involves activation of genes via proximal and distal regulatory regions. The activity of regulatory elements is determined by transcription factors (TFs) and epigenetic marks, but despite extensive mapping of such patterns, the extraction of regulatory principles remains challenging.ResultsHere we study differentially and similarly expressed genes along with their associated epigenomic profiles, chromatin accessibility and DNA methylation, during lineage specification at gastrulation in mice. Comparison of the three lineages allows us to identify genomic and epigenomic features that distinguish the two classes of genes.We show that differentially expressed genes are primarily regulated by distal elements, while similarly expressed genes are controlled by proximal housekeeping regulatory programs. Differentially expressed genes are relatively isolated within topologically associated domains, while similarly expressed genes tend to be located in gene clusters. Transcription of differentially expressed genes is associated with differentially open chromatin at distal elements including enhancers, while that of similarly expressed genes is associated with ubiquitously accessible chromatin at promoters.ConclusionBased on these associations of (linearly) distal genes’ transcription start sites (TSSs) and putative enhancers for developmental genes, our findings allow us to link putative enhancers to their target promoters and to infer lineage-specific repertoires of putative driver transcription factors, within which we define subgroups of pioneers and co-operators.

1690-P: Adipose Tissue Dectin-1 Upregulation in Hyperglycemic Obesity Involves TNF-α/NF-kB Mediated Signaling

Dectin-1 is a pathogen recognition receptor as well as an innate immune response modulator; its function in metabolic disorders is yet unclear. Previously, we identified dectin-1 as a biomarker of metabolic inflammation in obesity. In this study, we sought to identify potential signaling pathways that could modify the expression of the dectin-1 gene and assess the expression of dectin-1 in the adipose tissue (AT) of obese patients based on their diabetic status. The study cohort included 95 obese individuals split into two groups: prediabetics with moderate glycemia (Hb1Ac 6.5%, n = 49) and diabetics with hyperglycemia (Hb1Ac 6.5%, n = 46). Dectin-1 expression was assessed using immunohistochemistry. Gene expression and inflammatory markers were determined via qRT-PCR. We found a significant positive correlation between dectin-1 expression and HbA1C levels in AT isolated from obese individuals with HbA1C levels of 6.5% or higher. Dectin-1 gene expression was significantly correlated with several inflammatory markers; however, glycemic-dependent associations were also observed. Dectin-1 and TNF-α were found to be significantly correlated in AT from individuals with Hb1Ac 6.5%, indicating a possible mechanism of gene regulation between these two factors. As a result, we investigated the observed dectin-1/TNF-α crosstalk using in vitro cell culture and animal studies. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of dectin-1 gene and protein expression in their AT, which were restored by injecting exogenous TNF-α. ChIP studies showed that TNF-α induced dectin-1 gene transcription by mediating NF-kB binding to newly identified regulatory elements located in the dectin-1 proximal regulatory region. The interplay between dectin-1 and TNF-α signaling pathways is intriguing and has the potential to be a therapeutic target in obesity and diabetes. Furthermore, dectin-1 could be a potential marker for the onset of hyperglycemia and diabetes. Disclosure A.Al madhoun: None. D.Haddad: None. S.P.Kochumon: None. F.Alrashed: None. R.S.Thomas: None. L.P.Miranda: None. S.T.K.Sindhu: None. R.Ahmad: None. F.Almulla: None.

Adipose Tissue Caveolin-1 Upregulation in Obesity Involves TNF-α/NF-κB Mediated Signaling

Obesity is characterized by chronic low-grade inflammation. Obese people have higher levels of caveolin-1 (CAV1), a structural and functional protein present in adipose tissues (ATs). We aimed to define the inflammatory mediators that influence CAV1 gene regulation and the associated mechanisms in obesity. Using subcutaneous AT from 27 (7 lean and 20 obese) normoglycemic individuals, in vitro human adipocyte models, and in vivo mice models, we found elevated CAV1 expression in obese AT and a positive correlation between the gene expression of CAV1, tumor necrosis factor-alpha (TNF-α), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CAV1 gene expression was associated with proinflammatory cytokines and chemokines and their cognate receptors (r ≥ 0.447, p ≤ 0.030), but not with anti-inflammatory markers. CAV1 expression was correlated with CD163, indicating a prospective role for CAV1 in the adipose inflammatory microenvironment. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of CAV1 mRNA/proteins, which were elevated by administering exogenous TNF-α. Mechanistically, TNF-α induces CAV1 gene transcription by mediating NF-κB binding to its two regulatory elements located in the CAV1 proximal regulatory region. The interplay between CAV1 and the TNF-α signaling pathway is intriguing and has potential as a target for therapeutic interventions in obesity and metabolic syndromes.

Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis.

HighlightsThe conversion of 5-methylcytosine to 5-hydroxymethylcytosine relaxes the repressive marks of gene expression, and their imbalance leads to cancer development.Global loss of 5-hmC is found to be associated with the downregulation of TET1 and TET3 genes in breast cancer.Genome-wide analysis revealed 4809 differentially methylated regions and 4841 differentially hydroxymethylated regions in breast cancer.The abundance of 5-mC was observed at gene promoter regions while the 5-hmC was profusely distributed at the distal regulatory regions of the breast cancer genome. The accumulation of 5-hmC at distal regulatory sites can potentially enhance gene transcription.Alteration in the 5-hmC levels was positively associated with the respective gene expression. Novel 5-hmC candidates such as TXNL1, CNIH3, BNIPL, and CHODL were found to be promising diagnostic and therapeutic markers for breast cancer.An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>−5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

MIR205HG/LEADR Long Noncoding RNA Binds to Primed Proximal Regulatory Regions in Prostate Basal Cells Through a Triplex- and Alu-Mediated Mechanism.

Aside serving as host gene for miR-205, MIR205HG transcribes for a chromatin-associated long noncoding RNA (lncRNA) able to restrain the differentiation of prostate basal cells, thus being reannotated as LEADR (Long Epithelial Alu-interacting Differentiation-related RNA). We previously showed the presence of Alu sequences in the promoters of genes modulated upon MIR205HG/LEADR manipulation. Notably, an Alu element also spans the first and second exons of MIR205HG/LEADR, suggesting its possible involvement in target selection/binding. Here, we performed ChIRP-seq to map MIR205HG/LEADR chromatin occupancy at genome-wide level in prostate basal cells. Our results confirmed preferential binding to regions proximal to gene transcription start site (TSS). Moreover, enrichment of triplex-forming sequences was found upstream of MIR205HG/LEADR-bound genes, peaking at −1,500/−500 bp from TSS. Triplexes formed with one or two putative DNA binding sites within MIR205HG/LEADR sequence, located just upstream of the Alu element. Notably, triplex-forming regions of bound genes were themselves enriched in Alu elements. These data suggest, from one side, that triplex formation may be the prevalent mechanism by which MIR205HG/LEADR selects and physically interacts with target DNA, from the other that direct or protein-mediated Alu (RNA)/Alu (DNA) interaction may represent a further functional requirement. We also found that triplex-forming regions were enriched in specific histone modifications, including H3K4me1 in the absence of H3K27ac, H3K4me3 and H3K27me3, indicating that in prostate basal cells MIR205HG/LEADR may preferentially bind to primed proximal regulatory elements. This may underscore the need for basal cells to keep MIR205HG/LEADR target genes repressed but, at the same time, responsive to differentiation cues.

Phenotypic and Methylome Responses to Salt Stress in Arabidopsis thaliana Natural Accessions.

Salt stress threatens plant growth, development and crop yields, and has become a critical global environmental issue. Increasing evidence has suggested that the epigenetic mechanism such as DNA methylation can mediate plant response to salt stress through transcriptional regulation and transposable element (TE) silencing. However, studies exploring genome-wide methylation dynamics under salt stress remain limited, in particular, for studies on multiple genotypes. Here, we adopted four natural accessions of the model species Arabidopsis thaliana and investigated the phenotypic and genome-wide methylation responses to salt stress through whole-genome bisulfite sequencing (WGBS). We found that salt stress significantly changed plant phenotypes, including plant height, rosette diameter, fruit number, and aboveground biomass, and the change in biomass tended to depend on accessions. Methylation analysis revealed that genome-wide methylation patterns depended primarily on accessions, and salt stress caused significant methylation changes in ∼ 0.1% cytosines over the genomes. About 33.5% of these salt-induced differential methylated cytosines (DMCs) were located to transposable elements (TEs). These salt-induced DMCs were mainly hypermethylated and accession-specific. TEs annotated to have DMCs (DMC-TEs) across accessions were found mostly belonged to the superfamily of Gypsy, a type II transposon, indicating a convergent DMC dynamic on TEs across different genetic backgrounds. Moreover, 8.0% of salt-induced DMCs were located in gene bodies and their proximal regulatory regions. These DMCs were also accession-specific, and genes annotated to have DMCs (DMC-genes) appeared to be more accession-specific than DMC-TEs. Intriguingly, both accession-specific DMC-genes and DMC-genes shared by multiple accessions were enriched in similar functions, including methylation, gene silencing, chemical homeostasis, polysaccharide catabolic process, and pathways relating to shifts between vegetative growth and reproduction. These results indicate that, across different genetic backgrounds, methylation changes may have convergent functions in post-transcriptional, physiological, and phenotypic modulation under salt stress. These convergent methylation dynamics across accession may be autonomous from genetic variation or due to convergent genetic changes, which requires further exploration. Our study provides a more comprehensive picture of genome-wide methylation dynamics under salt stress, and highlights the importance of exploring stress response mechanisms from diverse genetic backgrounds.

Probabilistic inference of the genetic architecture underlying functional enrichment of complex traits

We develop a Bayesian model (BayesRR-RC) that provides robust SNP-heritability estimation, an alternative to marker discovery, and accurate genomic prediction, taking 22 seconds per iteration to estimate 8.4 million SNP-effects and 78 SNP-heritability parameters in the UK Biobank. We find that only ≤10% of the genetic variation captured for height, body mass index, cardiovascular disease, and type 2 diabetes is attributable to proximal regulatory regions within 10kb upstream of genes, while 12-25% is attributed to coding regions, 32–44% to introns, and 22-28% to distal 10-500kb upstream regions. Up to 24% of all cis and coding regions of each chromosome are associated with each trait, with over 3,100 independent exonic and intronic regions and over 5,400 independent regulatory regions having ≥95% probability of contributing ≥0.001% to the genetic variance of these four traits. Our open-source software (GMRM) provides a scalable alternative to current approaches for biobank data.

Reducing embryonic mtDNA copy number alters epigenetic profile of key hepatic lipolytic genes and causes abnormal lipid accumulation in adult mice.

Adverse fetal environment, in particular a shortage or excess of nutrients, is associated with increased risks of metabolic diseases later in life. However, the molecular mechanisms underlying this developmental origin of adult diseases remain unclear. Here, we directly tested the role of mitochondrial stress in mediating fetal programming in mice by enzymatically depleting mtDNA in zygotes. mtDNA-targeted plasmid microinjection is used to reduce embryonic mtDNA copy number directly, followed by embryo transfer. Mice with reduced zygote mtDNA copy number were born morphologically normal and showed no accelerated body weight gain. However, at 5 months of age these mice showed markedly increased hepatic lipidosis and became glucose-intolerant. Hepatic mRNA and protein expressions of peroxisome proliferator-activated receptor α (Pparα), a key transcriptional regulator of lipid metabolism, were significantly decreased as a result of increased DNA methylation in its proximal regulatory region. These results indicate that perturbation of mitochondrial function around the periconceptional period causes hypermethylation and thus suppressed expression of PPARα in fetal liver, leading to impaired hepatic lipid metabolism. Our findings provide the first direct evidence that mitochondrial stress mediates epigenetic changes associated with fetal programming of adult diseases in a mammalian system.

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Abstract 4917: Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway

Abstract Background: Hotspot estrogen receptor-α (ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer, with Y537S and D538G as the two most frequent hotspot mutations. A mostly unanswered question is and how these mutations facilitate metastatic processes. Methods: Frequencies of different ESR1 hotspot mutations were compared within three publicly available cohorts. Y537S and D538G genome-edited MCF7 and T47D cell models were used for in vitro characterization. Wound scratching, spheroid collective migration, chemotaxis assays were applied to examine different metastatic properties. Tail vein injection in nude mice followed by human CK19 lung section immune staining was used to characterize in vivo metastasis. Canonical Wnt pathway activity was studied using top-flash reporter assays, immunoblotting, and overexpression of dominant negative TCF4. ChIP-seq and ATAC-seq were utilized to profile ER global binding patterns and chromatin accessibility in cell models, respectively. Porcupine inhibitor-LGK974 was used to evaluate the efficiency of Wnt-targeted therapy. Results: D538G mutations were more frequent in ER+ metastatic lesions compared to Y537S among three metastatic breast cancer cohorts with unbiased SNV detection. In line with this, T47D-D538G ESR1 mutant cells showed strongly enhanced cell migration in vitro which was not observed in Y537S cells. We also observed increased lung micro-metastasis in vivo after tail vein injection of the D538G cells. Cell line transcriptomic analysis revealed uniquely hyperactivated Wnt pathway in D538G mutant cells, which was further confirmed in vitro by top-flash reporter and immunoblot. Suppression of canonical Wnt pathways blocked T47D-D538G specific cell migration. Combination treatment of fulvestrant and LGK974 synergistically inhibited D538G specific migration. Mechanistically, multiple Wnt regulator genes were found uniquely upregulated in D538G cells. Interestingly, none of these targets gained ER binding peaks at proximal regulatory regions, suggesting potential epigenetic regulation, which was confirmed in ATAC-seq results. Knockdown of FOXA1, a well-characterized pioneer factor, decreased canonical Wnt activity and abrogated the D538G-unique cell migration in short-term. Conclusion: T47D-D538G cells showed uniquely enhanced cell migration via Wnt hyperactivation, potentially mediated via epigenetic remodeling. These findings suggest the further study of potential combinatorial targeting of Wnt and ER signaling in D538G ESR1 mutant tumors. Citation Format: Zheqi Li, Kevin M. Levine, Spencer Arneson, Kristofer C. Berrett, Nolan M. Priedigkeit, Amir Bahreini, Jian Chen, Li Zhu, Jason S. Carroll, George C. Tseng, Peter C. Lucas, Jennifer M. Atkinson, Jason Gertz, Adrian V. Lee, Steffi Oesterreich. Estrogen receptor D538G mutation promotes cell migration via hyperactivation of Wnt signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4917.

Folate Receptor β (FRβ) Expression in Tissue-Resident and Tumor-Associated Macrophages Associates with and Depends on the Expression of PU.1.

As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor β (FRβ, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the “anti-inflammatory gene set”, which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRβ marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.

Transcriptional activity of FIGLA, NEUROG2, and EGR1 transcription factors associated with polymorphisms in the proximal regulatory region of GPR54 gene in cattle

Activity of transcription factors affect synthesis of G-protein coupled receptor 54 (GPR54), an important factor in regulation of initiation of puberty. Expression of the GPR54 gene in cattle is associated with polymorphisms in the proximal regulatory region (PRR) of the GPR54 gene. Transcription resulting in production of GPR54 mRNA transcript occurs as a result of transcription factor (TF) interactions in the PRR. Polymorphisms in the PRR may be associated with extent of activity of these TFs. Folliculogenesis-specific BHLH TF (FIGLA), neurogenin 2 (NEUROG2), and early growth response 1 (EGR1) are important in modulation of ovarian follicle development and neurons synthesizing GnRH, thus, regulating biosynthesis of luteinizing hormone. The aim of this study, therefore, was to assess the transcription-activating potential of binding sites for FIGLA, NEUROG2, and EGR1 TFs in the GPR54 promoter of cattle. Two luciferase-based promoters, ATC and CCT, which contain three single nucleotide polymorphisms (SNPs), A/C-794, T/C-663, and C/T-601, in the GPR54 PRR, were analyzed to evaluate gene expression and activation of different promoters by FIGLA, NEUROG2, and EGR1. The FIGLA induced GPR54 transcription through the CCT, whereas NEUROG2 and EGR1 induced GPR54 transcription through the ATC promoter-binding site. The CCT-activating effects of FIGLA were greater (2.56-fold) than the ATC-activating effects (P < 0.05). The ATC-activating effects of NEUROG2 and EGR1 were markedly greater (12.91- and 8.41-fold; P < 0.01) than CCT-activating effects. The polymorphisms, CCT and ATC, of the cattle GPR54 affect the activity of transcription factors, therefore, have an important effect on production of GPR54 mRNA transcript.

The SNPs in myoD gene from normal muscle developing individuals have no effect on muscle mass

BackgroundMyogenic Differentiation 1 (MyoD) is a crucial master switch in regulating muscle-specific gene transcription. Forced expression of myoD is equipped to induce several cell lineages into myoblast, which then differentiate and fuse into myotube. Pig is one of the most significant livestock supplying meat, and has been classified into lean, fat and miniature pig breeds. However, the mechanisms underlying muscle mass variation among different pig breeds have remained unclear. Considering the important effect of MyoD on muscle development, it remains to be investigated whether the difference in muscle mass is caused by its single nucleotide polymorphisms (SNPs) which are the major differences among pig breeds at DNA level.ResultsIn this study, we identified the locations of porcine myoD regulatory regions including proximal regulatory region (PRR), distal regulatory region (DRR), and core enhancer (CE) region. There are 8 SNPs in the regulatory regions and 6 SNPs in gene body region, which were identified from lean, fat and miniature pig populations. However, these SNPs have no effects on its temporal expression and transcriptional activity which might lead to the distinction in postnatal muscle mass. In addition, overexpression of myoD clones across from amphibious to mammals including xenopus tropicalis, chicken, mouse and pig whose gene identities vary from 68 to 84%, could promote myogenesis in NIH3T3 fibroblasts cells.ConclusionsThese results proved that myoD nucleotide variations from different pig populations have no effect on muscle mass, suggesting that the function of myoD is highly conserved not only among different pig breeds, but also across different species. Thus, it would be futile to discover SNPs affecting muscle mass in pig populations with normal muscle development.

Long-range interactions between proximal and distal regulatory regions in maize

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal–distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize.

MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma.

Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300-MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300-MITF-FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. SIGNIFICANCE: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.

Characterization of the Regulation and Function of Family with Sequence Similarity 83D (Fam83d) in Skeletal Muscle

Skeletal muscle atrophy is a serious condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d was analyzed by cloning fragments of the proximal promoter region located immediately upstream from the start of transcription and fusing them with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Analysis of the promoter of Fam83d revealed conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. To assess where Fam83d is localized in the cell, we fused Fam83d with green fluorescent protein (GFP), transfected the Fam83d‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and observed that Fam83d is localized exclusively to the cytoplasm. It has recently been determined that Fam83d contains a putative phospholipase domain, is mis‐regulated in several different cancer types, and may modulate MAP Kinase signaling. Therefore, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation and MAP Kinase signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK were dramatically repressed. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins.

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems.