c-Myc Enhancer Research Articles

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

Repetitive DNA symmetry elements negatively regulate gene expression in embryonic stem cells

Transcription factor (TF) binding to genomic DNA elements constitutes one of the key mechanisms that regulates gene expression program in cells. Both consensus and nonconsensus DNA sequence elements influence the recognition specificity of TFs. Based on the analysis of experimentally determined c-Myc binding preferences to genomic DNA, here we statistically predict that certain repetitive, nonconsensus DNA symmetry elements can relatively reduce TF-DNA binding preferences. This is in contrast to a different set of repetitive, nonconsensus symmetry elements that can increase the strength of TF-DNA binding. Using c-Myc enhancer reporter system containing consensus motif flanked by nonconsensus sequences in embryonic stem cells, we directly demonstrate that the enrichment in such negatively regulating repetitive symmetry elements is sufficient to reduce the gene expression level compared with native genomic sequences. Negatively regulating repetitive symmetry elements around consensus c-Myc motif and DNA sequences containing consensus c-Myc motif flanked by entirely randomized sequences show similar expression baseline. A possible explanation for this observation is that rather than complete repression, negatively regulating repetitive symmetry elements play a regulatory role in fine-tuning the reduction of gene expression, most probably by binding TFs other than c-Myc.

Abstract 394: Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract In the past decade, genome-wide association studies have identified a large number of genomic variants, specifically single-nucleotide polymorphisms (SNPs), associated with increased cancer risk. The molecular mechanisms underlying the vast majority of these associations remain largely unknown. Precise cellular models containing the variant of interest could be a crucial tool for establishing disease-relevant function in order to translate these discoveries into clinical application. CRISPR/Cas9 gene editing has provided a convenient and flexible method for creating isogenic cell lines via the homology-directed repair (HDR) pathway; however, methods for efficient generation and subsequent isolation of precisely edited cells have proven both expensive and time consuming. In response to this need, we have combined CRISPR/Cas9 HDR gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer risk variants in ~1 month without selectable markers or specialized methods such as digital droplet PCR or NGS. Utilizing this technology, we have been able to create cell lines differing by only a single base to model risk-associated SNPs located in the 8q24 risk locus in multiple cell types. The 8q24 risk locus is associated with increased risk for colorectal, breast, prostate, and, more recently, thyroid cancer. In this study, we use engineered isogenic cell lines modeling functional risk SNP rs6983267 in each of these cancer types to identify tissue-specific mechanisms of this multicancer risk locus. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in the pathogenesis of many cancers. However, how or if this mechanism varies between the different cancers this locus has been associated with remains uncertain. From preliminary studies, we do observe cell type variation in the regulatory effect of rs6983267. Not only does there seem to be a varying effect of the risk allele on c-Myc expression across the different cell types, but also risk allele enhancement of binding of the TCF7L2 transcription factor to the c-Myc enhancer rs6983267 is located within. Interestingly, rs6983267's regulatory effect seems most significant between homozygous reference cells and heterozygous cells within the same tissue type. We plan to delve further into these differences using RNA-seq and 4C-seq to assess whole transcriptomic and chromosomal interaction changes with changes in risk status. This study is one of first to compare risk mechanisms across different cancer tissue types; using precisely edited cellular models, we are able to detect cell-type specific variations in functional risk SNP effects on cancer development. Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 394.

Abstract 1445: Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract Colorectal Cancer (CRC) is one of the leading causes of mortality in the US, being the third most common cancer among both men and women. While an exact cause of CRC has not yet been elucidated, the existence of a clear heritable genetic component has been established. With the recent surge of data from Genome-Wide Association Studies, a handful of CRC-associated loci have been identified, each containing numerous Single Nucleotide Polymorphisms (SNPs). We hypothesize that among the ~25 loci associated with increased risk for CRC, there are driver SNPs functioning to contribute to initiation or progression of CRC development. It is crucial, in order to enhance our understanding CRC tumorigenesis, to extract these functional variants from the pool of associated SNPs and validate their causative significance within the cell. To model these functional SNPs, we have developed a novel “scarless” isolation pipeline for selecting isogenic clones with the desired single-base edit without selection markers following CRISPR-Cas9 mediated HDR. With this pipeline, we have successfully modeled our first selected SNP, rs6983267, from the 8q24 risk locus in HCT-116 cells, producing the heterozygous genotype (G/T) from the parental homozygous risk (G/G) genotype. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in CRC pathogenesis. In line with previous studies, we observe a slight decrease in c-Myc expression in our heterozygous clones compared to wildtype homozygous risk. Moreover, we observe a preferential binding of the transcription factor in control of c-Myc transcriptional regulation, TCF7L2, to the risk allele over the reference allele while binding of proximal factors such as CTCF show no allelic preference. Interestingly, we also see significant changes in expression of target genes of the WNT signaling pathway. Dysregulation of WNT is a hallmark characteristic of CRC. These changes indicate there is indeed an effect of the risk allele of rs6983267 on overall WNT activity in CRC cells due to increased binding of transcription factor TCF7L2. This novel SNP editing pipeline provides a method to study precise effects of single base changes located anywhere in the genome. Utilizing it to model other risk SNPs will enable the enhancement of our current understanding of GWAS-identified risk loci in CRC pathogenesis at the molecular level. Citation Format: Nicole Coggins, Jacob Stultz, David Segal, Luis Carvajal-Carmona. Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2017-1445

Abstract LB-83: Transcriptional mechanisms of NR4A orphan nuclear receptor tumor suppression in AML

Abstract Acute myeloid leukemias are a highly heterogeneous group of diseases associated with corruption of myeloid cell differentiation, accumulation of immature blasts, and emergence of a transformed leukemia initiating cell (LIC) capable of sustaining leukemic expansion. Genetic and phenotypic properties of LICs are highly variable among AML patients as a consequence of variable cytogenetics, stem/progenitor cell of origin and stage of disease progression. The orphan nuclear receptors NR4A1 and NR4A3 are potent AML tumor suppressors whose deletion in mice leads to rapid AML development. They are also silenced in cytogenetically diverse human LICs, and their anti-leukemic effect is associated with acute transcriptional repression of c-MYC and reprogramming of a core MYC oncogenic gene signature that is common to LICs of distinct cytogenetic backgrounds. c-MYC transcription is controlled at the step of transcriptional elongation, and high levels of c-MYC expression are maintained in many cancers, including AML, by establishment of cancer cell-selective super enhancers that are marked by high levels of the BET bromodomain protein, BRD4, a key regulator of c-MYC transcription elongation together with BRD4 associated components of the Mediator coactivator complex. BRD4 promotes transcription elongation of c-MYC by recruiting the positive transcription elongation factor, P-TEFb, a catalytic component of the super elongation complex (SEC) which phosphorylates RNA PolII at serine 2 to enable release of promoter paused RNA PolII. Here we investigated the transcriptional mechanisms of NR4A1-dependent c-MYC repression. Interrogation of genome-wide NR4A1 binding in Kasumi-1 AML-ETO positive human AML cells revealed NR4A1 occupancy within the 5’ proximal regions of the c-MYC gene and at a downstream enhancer region previously identified as a c-MYC super enhancer in the RN2 mouse model of MLL-AF9/NrasG12D driven AML. Consistent with its classification as a super enhancer, we found strong enrichment of BRD4 and the MED1 component of the Mediator complex at the c-MYC enhancer in Kasumi-1 AML cells in the absence of NR4A1. Interrogation of the effects of acute rescue of NR4A1 expression on the enhancer status of this region and transcriptional elongation of c-MYC revealed that NR4A1 represses c-MYC transcription elongation by interacting directly with the enhancer locus to inhibit recruitment of Mediator complex and BRD4, leading to loss of P-TEFb recruitment to both the enhancer and c-MYC gene and a loss of elongation competent RNA PolII. Together, these data indicate the NR4A1 negatively regulates transcriptional elongation at the c-MYC locus by interfering with factors required for super enhancer maintenance and AML selective overexpression of c-MYC. Since AML cells which rely on c-MYC overexpression are extremely sensitive to BRD4 inhibition, our results predict that strategies directed toward NR4A activation in AML cells may provide an alternative means of targeting c-MYC driven AMLs. Citation Format: Ryan P. Duren, Orla M. Conneely. Transcriptional mechanisms of NR4A orphan nuclear receptor tumor suppression in AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-83. doi:10.1158/1538-7445.AM2014-LB-83

ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

BackgroundInsulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.Methods and resultsThe expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.ConclusionsOur results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

Androgen receptor (AR) suppresses normal human prostate epithelial cell proliferation via AR/β-catenin/TCF-4 complex inhibition of c-MYC transcription.

Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs). Lentiviral shRNA stable knock down of c-MYC, β-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent β-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear β-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/β-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain. Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of β-catenin/TCF-4 complexes at both 5' and 3' c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/β-catenin/TCF-4 complexes, which retains binding to 3' c-MYC enhancer, but now suppresses c-MYC transcription.

Substance P activates the Wnt signal transduction pathway and enhances the differentiation of mouse preosteoblastic MC3T3-E1 cells.

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10−10 to 10−8 M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10−9 to 10−8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10−8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10−8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.

Protein Kinase C Delta Negatively Modulates Canonical Wnt Pathway and Cell Proliferation in Colon Tumor Cell Lines

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional β-catenin destruction complex, did not affect β-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal β-catenin destruction complex, negatively affected β-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of β-catenin degradation, because PKCδ inhibition induces β-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.

In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP‐dependent protein kinase, and its relevance for biochemical activities

The zinc-finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single-stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis. CNBP cDNAs from a number of vertebrates have been cloned and analysed. CNBP is mainly conformed by seven retroviral Cys-Cys-His-Cys zinc-knuckles and a glycine/arginine rich region box. CNBP amino acid sequences show a putative Pro-Glu-Ser-Thr site of proteolysis and several putative phosphorylation sites. In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities. We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts. In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid-blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at 5 days postfertilization. The cAMP-dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site. Site-directed mutagenesis replacing the PKA phospho-acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D. rerio but also in other vertebrates. CNBP phosphorylation does not change single-stranded nucleic acid binding capability. Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c-myc) promoter, an element responsible for c-myc enhancer transcription. Our results suggest that phosphorylation might be a conserved post-translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c-myc, during vertebrate embryogenesis.

The APC tumor suppressor counteracts β-catenin activation and H3K4 methylation at Wnt target genes

The APC tumor suppressor controls the stability and nuclear export of beta-catenin (beta-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. We show here that beta-cat and APC have opposing actions at Wnt target genes in vivo. The beta-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and we show that beta-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and we find that ubiquitin is necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that beta-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and betaTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, betaTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. We conclude that, in addition to regulating the stability of beta-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells.

Transcription and replication silencer element is present within conserved region of human Alu repeats interacting with nuclear protein

Human cells contain a nuclear protein interacting with Alu repeats, and this protein seems to recognize a conserved sequence motif, GGAGGC, present within the RNA polymerase III promoter and within the SV40 T-antigen-dependent ARS-like element. To study the potential functional role of this element, we have inserted the sequence into a chloramphenicolacetyltransferase (CAT) expression vector with a SV40 promoter and enhancer element from the up-stream region of the human c-myc gene, and transfected HeLa cells with the resulting plasmid. Analysis of expression by the CAT assay indicates that the Alu-derived sequence supresses transcription of the CAT gene driven by the c-myc enhancer/SV40 promoter. The Alu-derived sequence also inhibits ARS activity of the c-myc enhancer. The data allow the explaination of the transcriptional inactivity of Alu repeats in HeLa cells, and suggest the existence of a negative control of Alu transcription.