Identification of MYC intron 2 regions that modulate expression
Ontology highlight
ABSTRACT:
SUBMITTER: Tompkins V
PROVIDER: S-EPMC10795982 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The conserved transcription factor Myc regulates cell growth, proliferation and apoptosis, and its deregulation has been associated with human pathologies. Although specific miRNAs have been identified as fundamental components of the Myc tumorigenic program, how Myc regulates miRNA biogenesis remains controversial. Here we showed that Myc functions as an important regulator of miRNA biogenesis in Drosophila by influencing both miRNA gene expression and processing. Through the analysis of ChIP-Seq datasets, we discovered that nearly 56% of Drosophila miRNA genes show dMyc binding, exhibiting either the canonical or non-canonical E-box sequences within the peak region. Consistently, reduction of dMyc levels resulted in widespread downregulation of miRNAs gene expression. dMyc also modulates miRNA processing and activity by controlling Drosha and AGO1 levels through direct transcriptional regulation. By using in vivo miRNA activity sensors we demonstrated that dMyc promotes miRNA-mediated silencing in different tissues, including the wing primordium and the fat body. We also showed that dMyc-dependent expression of miR-305 in the fat body modulates Dmp53 levels depending on nutrient availability, having a profound impact on the ability of the organism to respond to nutrient stress. Indeed, dMyc depletion in the fat body resulted in extended survival to nutrient deprivation which was reverted by expression of either miR-305 or a dominant negative version of Dmp53. Our study reveals a previously unrecognized function of dMyc as an important regulator of miRNA biogenesis and suggests that Myc-dependent expression of specific miRNAs may have important tissue-specific functions.
| S-EPMC10491395 | biostudies-literature
Project description:Cystin is a novel cilia-associated protein that is disrupted in the cpk mouse, a well-characterized mouse model of autosomal recessive polycystic kidney disease (ARPKD). Interestingly, overexpression of the Myc gene is evident in animal models of ARPKD and is thought to contribute to the renal cystic phenotype. Using a yeast two-hybrid approach, the growth suppressor protein necdin, known to modulate Myc expression, was found as an interacting partner of cystin. Deletion mapping demonstrated that the C-terminus of cystin and both termini of necdin are required for their mutual interaction. Speculating that these two proteins may function to regulate gene expression, we developed a luciferase reporter assay and observed that necdin strongly activated the Myc P1 promoter, and cystin did so more modestly. Interestingly, the necdin effect was significantly abrogated when cystin was co-transfected. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed a physical interaction with both necdin and cystin and the Myc P1 promoter, as well as between these proteins. The data suggest that these proteins likely function in a regulatory complex. Thus, we speculate that Myc overexpression in the cpk kidney results from the dysregulation of the cystin-necdin regulatory complex and c-Myc, in turn, contributes to cystogenesis in the cpk mouse.
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Project description:The putative role of (TG/CA)n repeats in the regulation of transcription has recently been reported for several cancer- and disease-related genes, including the genes encoding the epidermal growth factor receptor (EGFR), hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2) and interferon-gamma (IFNG). These studies indicated a correlation between gene expression levels and the presence or length of (TG/CA)n repeats in their intron 1 regions. A genome-wide search for genes with similar features may provide evidence of whether these dinucleotide repeats represent a class of universal regulators of gene expression, which has recently begun to be investigated as a quantitative complex phenotype. Using a public database of simple repeats, we identified 330 genes containing potentially polymorphic long (TG/CA)n repeats (n >or= 12) in their intron 1 regions. One known physiological pathway, the calcium signalling pathway, was found to be enriched among the genes containing long repeats. In addition, certain biological processes, such as cation transport, signal transduction and ion transport, were found to be enriched in these genes. Genotyping of the long repeats showed that the majority of these dinucleotide repeats were polymorphic in the HapMap CEU (Caucasians from Utah, USA) samples of northern and western European ancestry. Evidence for a significant association between these repeats and gene expression was not observed in the genes selected based on their expression profiles in the HapMap CEU samples. Our current findings, therefore, do not support a role for these repeats as a class of universal gene expression regulators. A more comprehensive evaluation of the relationship between these repeats and gene expression, potentially in other tissues, may be necessary to illustrate their roles in gene regulation in the future.
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Project description:Osteosarcoma (OS) is the most common primary bone tumor of childhood. Approximately 20%-30% of OSs carry amplification of chromosome 8q24, which harbors the oncogene c-MYC and correlates with a poor prognosis. To understand the mechanisms that underlie the ability of MYC to alter both the tumor and its surrounding tumor microenvironment (TME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox-c-MycT58A p53fl/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc-knockin GEMM had rapid tumor development with a high incidence of metastasis. MYC-dependent gene signatures in our murine model demonstrated significant homology to the human hyperactivated MYC OS. We established that hyperactivation of MYC led to an immune-depleted TME in OS demonstrated by the reduced number of leukocytes, particularly macrophages. MYC hyperactivation led to the downregulation of macrophage colony-stimulating factor 1, through increased microRNA 17/20a expression, causing a reduction of macrophage population in the TME of OS. Furthermore, we developed cell lines from the GEMM tumors, including a degradation tag-MYC model system, which validated our MYC-dependent findings both in vitro and in vivo. Our studies utilized innovative and clinically relevant models to identify a potentially novel molecular mechanism through which MYC regulates the profile and function of the OS immune landscape.
| S-EPMC10371352 | biostudies-literature
Project description:RationaleThe growing recognition of the importance of splicing, together with rapidly accumulating RNA-sequencing data, demand robust high-throughput approaches, which efficiently analyze experimentally derived whole-transcriptome splice profiles.ResultsWe have developed a computational approach, called SNPlice, for identifying cis-acting, splice-modulating variants from RNA-seq datasets. SNPlice mines RNA-seq datasets to find reads that span single-nucleotide variant (SNV) loci and nearby splice junctions, assessing the co-occurrence of variants and molecules that remain unspliced at nearby exon-intron boundaries. Hence, SNPlice highlights variants preferentially occurring on intron-containing molecules, possibly resulting from altered splicing. To illustrate co-occurrence of variant nucleotide and exon-intron boundary, allele-specific sequencing was used. SNPlice results are generally consistent with splice-prediction tools, but also indicate splice-modulating elements missed by other algorithms. SNPlice can be applied to identify variants that correlate with unexpected splicing events, and to measure the splice-modulating potential of canonical splice-site SNVs.Availability and implementationSNPlice is freely available for download from https://code.google.com/p/snplice/ as a self-contained binary package for 64-bit Linux computers and as python source-code.Contactpmudvari@gwu.edu or horvatha@gwu.eduSupplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Molecular biotechnology has made it possible to explore the potential of plants for different purposes. The 3' regulatory regions have a great diversity of cis-regulatory elements directly involved in polyadenylation, stability, transport and mRNA translation, essential to achieve the desired levels of gene expression. A complex interaction between the cleavage and polyadenylation molecular complex and cis-elements determine the polyadenylation site, which may result in the choice of non-canonical sites, resulting in alternative polyadenylation events, involved in the regulation of more than 80% of the genes expressed in plants. In addition, after transcription, a wide array of RNA-binding proteins interacts with cis-acting elements located mainly in the 3' untranslated region, determining the fate of mRNAs in eukaryotic cells. Although a small number of 3' regulatory regions have been identified and validated so far, many studies have shown that plant 3' regulatory regions have a higher potential to regulate gene expression in plants compared to widely used 3' regulatory regions, such as NOS and OCS from Agrobacterium tumefaciens and 35S from cauliflower mosaic virus. In this review, we discuss the role of 3' regulatory regions in gene expression, and the superior potential that plant 3' regulatory regions have compared to NOS, OCS and 35S 3' regulatory regions.
| S-EPMC7457121 | biostudies-literature
Project description:Both OTX2 and MYC are important oncogenes in medulloblastoma, the most common malignant brain tumor in childhood. Much is known about MYC binding to promoter regions, but OTX2 binding is hardly investigated. We used ChIP-on-chip data to analyze the binding patterns of both transcription factors in D425 medulloblastoma cells. When combining the data for all promoter regions in the genome, OTX2 binding showed a remarkable bi-modal distribution pattern with peaks around -250 bp upstream and +650 bp downstream of the transcription start sites (TSSs). Indeed, 40.2% of all OTX2-bound TSSs had more than one significant OTX2-binding peak. This OTX2-binding pattern was very different from the TSS-centered single peak binding pattern observed for MYC and other known transcription factors. However, in individual promoter regions, OTX2 and MYC have a strong tendency to bind in proximity of each other. OTX2-binding sequences are depleted near TSSs in the genome, providing an explanation for the observed bi-modal distribution of OTX2 binding. This contrasts to the enrichment of E-box sequences at TSSs. Both OTX2 and MYC binding independently correlated with higher gene expression. Interestingly, genes of promoter regions with multiple OTX2 binding as well as MYC binding showed the highest expression levels in D425 cells and in primary medulloblastomas. Genes within this class of promoter regions were enriched for medulloblastoma and stem cell specific genes. Our data suggest an important functional interaction between OTX2 and MYC in regulating gene expression in medulloblastoma.
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Project description:Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.
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Project description:The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.
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Project description:The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small molecules.
| S-EPMC4719142 | biostudies-literature