Transcriptional Enhancer Element Research Articles

A Prox1 enhancer represses haematopoiesis in the lymphatic vasculature.

Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development1-3. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.

Genomic glucocorticoid action in embryonic mouse neural stem cells

Prenatal exposure to synthetic glucocorticoids (sGCs) reprograms brain development and predisposes the developing fetus towards potential adverse neurodevelopmental outcomes. Using a mouse model of sGC administration, previous studies show that these changes are accompanied by sexually dimorphic alterations in the transcriptome of neural stem and progenitor cells (NSPCs) derived from the embryonic telencephalon. Because cell type-specific gene expression profiles tightly regulate cell fate decisions and are controlled by a flexible landscape of chromatin domains upon which transcription factors and enhancer elements act, we multiplexed data from four genome-wide assays: RNA-seq, ATAC-seq (assay for transposase accessible chromatin followed by genome wide sequencing), dual cross-linking ChIP-seq (chromatin immunoprecipitation followed by genome wide sequencing), and microarray gene expression to identify novel relationships between gene regulation, chromatin structure, and genomic glucocorticoid receptor (GR) action in NSPCs. These data reveal that GR binds preferentially to predetermined regions of accessible chromatin to influence gene programming and cell fate decisions. In addition, we identify SOX2 as a transcription factor that impacts the genomic response of select GR target genes to sGCs (i.e., dexamethasone) in NSPCs.

Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.

Yy1 Depletion in Pancreatic Beta Cells Leads to Energy Source Switch From Glycolysis to Oxidative Phosphorylation

Background: Gene expression is determined by structural interactions in between transcription factors, cofactors and enhancer elements, as well as enhancer-promoter interactions (1). Both YY1 and CTCF are essential, zinc finger proteins that bind hypo-methylated DNA sequences, form homodimers, and thus facilitate DNA loop formation (1). ​However, YY1 preferentially occupies interacting enhancers and promoters, whereas CTCF preferentially occupies sites distal from these regulatory elements, forming larger loops and participating in insulation (1). A sequencing study of spontaneous functional insulinomas in a Chinese cohort identified a somatic a hotspot mutation in YY1 (c.C1115G/p.T372R) in 30% of the cases, associated with increased YY1 activity (2). YY1 is a critical transcription factor involved in the regulation of proliferation and metabolism (2). Hypothesis: YY1 loss-of-function alters energy source preference in pancreatic β-cells. Methods: YY1 stable loss-of-function in mouse insulinoma cell lines was achieved by shRNA lentiviral transduction. Mitochondrial membrane potential (MMP) was measured via flow cytometry of aggregated mitochondria to monomeric mitochondria ratio. Mitostress and complex-substrate controlled respiration were measured by Seahorse analyzer. Mitochondrial copy number was assessed by mitochondrial to nuclear DNA ratio. Quantitative qPCR and Western blotting were used to assess mitochondrial gene and protein expression. Results: Our data indicated that YY1 deficient β-cells showed increased MMP and maximal respiration. No significant differences were found in basal respiration, ATP production, proton leak, non-mitochondrial oxygen consumption or coupling efficiency. We also found that YY1 deficient β-cells exhibited reduced glycolytic capacity and decreased ETC complex IV activity, with concurrent increased complex I and II activity. In addition, YY1 deficient β-cells exhibited elevated mitochondrial copy number​ and increased quantitative mRNA of mitochondrial gene expression, which could be correlated with increased PGC1-α expression. Conclusions: YY1 is critical in the metabolic regulation of β-cells, particularly in the facilitation of glycolytic metabolism. YY1 activating mutations in functional spontaneous insulinoma cells can lead to a proliferation dysregulation accompanied by a metabolic switch that favors glycolysis, while the opposite occurs in YY1 deficient β-cells.

Stag2 Regulates Hematopoietic Differentiation and Self-Renewal through Alterations in Gene Expression and Topological Control

Cell-type specific transcriptional programs are regulated by the activity of tissue-specific transcription factors and enhancer elements within structurally defined topologically associating domains (TADs). The coordinated and dynamic changes in chromatin architecture are highly regulated as transcriptional output is influenced by chromatin accessibility, histone modification, promoter enhance interactions, and recruitment of transcriptional co-activator complexes. The genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias, Ewing sarcoma, and glioblastoma multiforme. Despite this, the mechanistic role of STAG2 in gene regulation, hematopoietic function, and tumor suppression has not been delineated. We show that somatic Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation across all three lineages consistent with myelodysplasia. Chromatin immunoprecipitation sequencing of Stag2-deficient HSPCs revealed that Stag2 and Stag1 have both shared and non-redundant cistromes with Stag1/2 common binding sites enriching at TAD boundaries with CTCF occupancy. This maintains TAD integrity in the setting of Stag2 loss of function which we confirmed with Hi-C chromosome capture. High resolution of the Hi-C data at 10kB resolution identified a specific role for Stag2, but not Stag1, in maintaining short-range chromatin interactions, specifically at genes with PU.1 and IRF8 motifs. While co-deletion of Stag2 and Stag1 resulted in synthetic lethality, Stag2 loss alone resulted in decreased chromatin accessibility and reduced transcriptional output at key PU.1 target loci involved in lineage-specification, including reduced Ebf1 and Pax5 expression resulting in impaired B-lineage differentiation. We investigated whether expression of PU.1 could overcome the non-permissive chromatin state at these key downstream targets and rescue hematopoietic differentiation; however, PU.1 expression could not restore Ebf1 expression or B cell differentiation and did not attenuate the serial replating capacity of Stag2 deficient hematopoietic stem/progenitor cells (HSPCs). Given this transcriptional "choke-point" we investigated whether expression of Ebf1 would have a more significant impact on Stag2 deficient cells. Indeed Ebf1 rescue restored B cell colony formation/differentiation in vitro and in vivo and abrogated serial replating of Stag2 deficient HSPCs. These data highlight the non-hierarchical and non-redundant relationship between transcription factors and chromatin architecture and demonstrate a key role for Stag2-regulated local interactions in transcription factor output and hematopoietic differentiation. Nonetheless, the mechanistic underpinnings of the structural basis for transcriptional regulation remain associative. We have recently been able to reduce the cell input for Hi-C assays such that we can now analyze the chromatin architecture of purified populations and model the structural transition from Lin- Sca-1+ Kit+ hematopoietic stem cells to committed granulocyte macrophage precursor cells both in normal hematopoiesis and in the Stag2 deficient setting. Previous studies using in vitro systems have shown that complete cohesin depletion results in the loss of structural loop components; however, cohesin levels are reduced, but not absent, in cancer cells. As such, our studies highlight a key role for locus-specific alterations in gene regulation and DNA interactions in Stag2 deficient cells, which results in altered gene expression and contributes to transformation. Taken together, these data identify a key role for Stag2 loss in transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation. Moreover, we illustrate a critical link between cohesin, chromosomal contacts, and gene regulation that contributes to hematopoietic transformation. Disclosures Viny: Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Dekker:Arima Genomics: Membership on an entity's Board of Directors or advisory committees. Levine:Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees.

Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-\u03baB

Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is identified as a novel cell surface marker for human T-cell leukemia virus (HTLV-1)-infected T cells. Adult T-cell leukemia/lymphoma (ATLL) is developed in HTLV-1-infected T-cells after a long infection period. To examine the mechanism of CADM1 overexpression in ATLL, we first identified that CADM1 is transcriptionally up-regulated by a transcriptional enhancer element through NF-κB signaling pathway. In HTLV-1-infected T-cells, CADM1 expression is dependent on HTLV-1/Tax through activation of canonical and non-canonical NF-κB; however, in ATLL cells with frequent loss of Tax expression, the activation of canonical NF-κB only enhances the CADM1 expression. Along with active mutations in signaling molecules under T-cell recepor (TCR) signaling, degradation of p47, a negative regulator of NF-κB, was essential for activation of canonical NF-κB through stabilization of NEMO (NF-κB essential modulator). The mechanism of p47 degradation is primarily dependent on activation of lysosomal-autophagy and the autophagy is activated in most of the HTLV-infected and ATLL cells, suggesting that the p47 degradation may be a first key molecular event during HTLV-1 infection to T-cells as a connector of two important signaling pathways, NF-κB and autophagy.

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.

Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells.

Human embryonic carcinoma (EC) cells are shown to restrict the expression of murine leukemia virus genomes but not retroviral genomes of the lentiviral or betaretroviral families. The block occurs at the level of transcription and is accompanied by the deposition of repressive histone marks and methylation of the integrated proviral DNA. The host machinery required for silencing in human EC cells is distinct from that in murine EC cell lines: the histone methyltransferase SETDB1 is required, but the widely utilized corepressor TRIM28/Kap1 is not. A transcriptional enhancer element from the Mason-Pfizer monkey virus can override the silencing and promote transcription of chimeric proviral DNAs. The findings reveal novel features of human EC gene regulation not present in their murine counterparts.

Association of Forced Vital Capacity with the Developmental Gene NCOR2.

BackgroundForced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes.MethodsPer-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 children (ALSPAC). Associations were considered replicated if the replication p-value survived Bonferroni correction (p<0.002; 0.05/25), with a nominal p-value considered as suggestive evidence. For SNPs with evidence of replication, effects on the expression levels of nearby genes in lung tissue were tested in 1,111 lung samples (Lung eQTL consortium), with further functional investigation performed using public epigenomic profiling data (ENCODE).ResultsNCOR2-rs12708369 showed strong replication in children (p = 0.0002), with replication unavailable in adults due to low imputation quality. This intronic variant is in a strong transcriptional enhancer element in lung fibroblasts, but its eQTL effects could not be tested due to low imputation quality in the eQTL dataset. SERPINE2-rs6754561 replicated at nominal level in both adults (p = 0.036) and children (p = 0.045), while WNT16-rs2707469 replicated at nominal level only in adults (p = 0.026). The eQTL analyses showed association of WNT16-rs2707469 with expression levels of the nearby gene CPED1. We found no statistically significant eQTL effects for SERPINE2-rs6754561.ConclusionsWe have identified a new gene, NCOR2, in the retinoic acid signalling pathway pointing to a role of vitamin A metabolism in the regulation of FVC. Our findings also support SERPINE2, a COPD gene with weak previous evidence of association with FVC, and suggest WNT16 as a further promising candidate.

Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2.

Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers.

BackgroundThe AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers.ResultsTransgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5′ sequence and 919 bp of 3′ sequence (AIL6:cAIL6-3′) fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5′ and 3′ sequence (AIL6:gAIL6-3′) can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5′ sequence and 919 bp of 3′ sequence (AIL6m:gAIL6-3′) complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3′ and AIL6m:gAIL6-3′ lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation.ConclusionsThe results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.

Functional FLT1 Genetic Variation is a Prognostic Factor for Recurrence in Stage I–III Non–Small-Cell Lung Cancer

We propose that single-nucleotide polymorphisms (SNPs) in genes of the vascular endothelial growth factor pathway of angiogenesis will associate with survival in non-small-cell lung cancer (NSCLC) patients. Fifty-three SNPs in vascular endothelial growth factor-pathway genes were genotyped in 150 European stage I-III NSCLC patients and tested for associations with patient survival. Replication was performed in an independent cohort of 142 European stage I-III patients. Reporter gene assays were used to assess the effects of SNPs on transcriptional activity. In the initial cohort, five SNPs associated (q < 0.05) with relapse-free survival (RFS). The minor alleles of intronic FLT1 SNPs, rs7996030 and rs9582036, associated with reduced RFS (hazard ratio [HR] = 1.67 [95% confidence interval, CI, 1.22-2.29] and HR = 1.51 [95% CI, 1.14-2.01], respectively) and reduced transcriptional activity. The minor alleles of intronic KRAS SNPs, rs12813551 and rs10505980, associated with increased RFS (HR = 0.64 [0.46-0.87] and HR = 0.64 [0.47-0.87], respectively), and the minor allelic variant of rs12813551 also reduced transcriptional activity. Lastly, the minor allele of the intronic KRAS SNP rs10842513 associated with reduced RFS (HR = 1.65 [95% CI, 1.16-2.37]). Analysis of the functional variants suggests they are located in transcriptional enhancer elements. The negative effect of rs9582036 on RFS was confirmed in the replication cohort (HR = 1.69 [0.99-2.89], p = 0.028), and the association was significant in pooled analysis of both cohorts (HR = 1.67 [1.21-2.30], p = 0.0001). The functional FLT1 variant rs9582036 is a prognostic determinant of recurrence in stage I-III NSCLC. Its predictive value should be tested in the adjuvant setting of stage I-III NSCLC.

A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Thymidylate synthase polymorphism in sporadic colorectal and gastric cancer in Tunisian population: a predictive role in 5-fluorouracil based chemotherapy treatment

In our study, we investigate the possible association of thymidylate synthase polymorphism, 28bp tandem repeat in 5'-UTR (transcription enhancer element) with susceptibility of colorectal and gastric cancer in Tunisian population. Because thymidylate synthase provides an effective prediction of chemotherapy treatment based on 5-fluorouracil, our interest in this study was focused on finding an eventual interaction between thymidylate synthase polymorphism and treatment of sporadic colorectal and gastric cancer. Whole blood was collected into EDTA tube, after centrifugation for 15min, the buffy coat was isolated, and genotyping of TS 5'-UTR polymorphism was carried by polymerase chain reaction method using appropriate primers. Determination of the different genotypes was done directly on the stained agarose gel. Our finding showed that the 5'tandem repeat polymorphism of the thymidylate synthase gene is associated with risk of colorectal cancer; thus, LL (3R/3R) genotype is significantly high in patients with colorectal cancer compared to controls (P=0.002; OR 2.7; 95% CI 1.4-5.2). In addition, we found a positive association between SL (2R/3R) genotype in the thymidylate synthase 5'-UTR and gastric cancer risk (P=0.015; OR 4.46; 95% CI 1.08-19.64). Furthermore, we found a correlation of thymidylate synthase polymorphism with the fluorouracil-based therapy regimes and also with preoperatory radiation in patients with colorectal cancer. Thymidylate synthase is associated with risk of colorectal cancer but not with gastric cancer; however, heterozygous SL (2R/3R) polymorphism is associated with risk of gastric cancer; moreover, the 5' tandem repeat polymorphism of thymidylate synthase gene was an independent predictor of the clinical treatment.

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

Asparagine synthetase (ASNS) gene polymorphism is associated with the outcome of childhood acute lymphoblastic leukemia by affecting early response to treatment

The polymorphism of 14-bp tandem repeat sequence located in the ASNS gene probably acts as a transcriptional enhancer element and leads to higher expression of the gene in carriers of more than 2 repeats (>R2). We searched for an association with disease outcome in 264 children with ALL. A multivariate proportional hazard regression model adjusted for age at diagnosis (HR (95%CI)=1.05 (1.04-1.09)) and high-risk group (HR(95%CI)=3.47 (1.74-6.88)) revealed that R3 carriers with a poor response at day 15 had an increased risk of events, HR (95%CI)=2.72 (1.06-6.96). These results suggest a conditional interaction between the ASNS polymorphism and an early response to chemotherapy among pediatric patients with ALL.

Functional Variants at the 11q13 Risk Locus for Breast Cancer Regulate Cyclin D1 Expression through Long-Range Enhancers

Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.

Abstract 9127: C-myb-regulated Expression Of Mir-143/145 In Embryonic Stem Cell-derived And Adult Vascular Smooth Muscle Cells

Background: The c-myb gene encodes for the transcription factor c-Myb, which has been implicated in the differentiation of vascular smooth muscle cells (VSMCs) from neural crest and embryonic stem cells (ESCs). Mouse ESCs lacking c-myb (KO) failed to express SMC-specific genes and did not differentiate into contractile SMCs in embryoid bodies (EB), with c-Myb regulating the formation of a specific Flk-1+/PDGFRα- (F+/P-) progenitor of VSMCs. Others showed that expression of the miR-143/145 cluster is restricted to adult SMC lineages and promotes the expression of contractile genes. Hypothesis: c-Myb mediates the transcriptional regulation of miR-143/145. Methods &amp;Results : Flow-sorting of cardiovascular-directed d3.75 EBs showed high levels of miR-143/145 expression in F+/P- (c-myb high) cells, with low levels in other (c-myb low: F+/P+ and F-/P+) cell populations. Importantly, no differences in the expression levels of miR-143/145 could be detected in F+/P-, F-/P+ and F+/P+ cells derived from c-myb KO EBs. Multiple putative c-Myb consensus-binding sites (conserved in human and mouse) were identified in 3kb and 20kb enhancers upstream of the miR-143/145 cluster. Chromatin immunoprecipitation assays in both cardiovascular-directed d3.75 EBs and immortalized mouse adult aortic SMCs (MOVAS) revealed c-Myb binding to multiple specific sites. Next, c-Myb responsiveness of wild-type and deletion mutant enhancer-reporter constructs was assayed in HEK293 cells. These studies confirmed two c-Myb-dependent miR-143/145 enhancer activities. Conclusion: Our data suggest that c-Myb-regulated VSMC differentiation and contractile gene expression may be mediated by direct binding to specific transcription enhancer elements in the miR-143/145 cluster. *Authors have contributed equally to this work.

Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1.

The hereditary mixed polyposis syndrome (HMPS) was first described about 50 years ago in a large Ashkenazi Jewish family from St Mark’s Hospital, London. The family showed apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma in a high proportion of individuals. In the last 15 years, we have mapped the HMPS gene to chromosome 15q13.3 and identified an ancestral haplotype common to all the known HMPS families. Here, we have used genetic mapping, copy number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a large duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This mutation has no effect on SCG5 expression, but is associated with greatly increased, allele-specific GREM1 expression. Whilst GREM1 is expressed in intestinal sub-epithelial myofibroblasts in controls, HMPS patients predominantly express GREM1 in the epithelium of the large bowel. The HMPS duplication contains predicted transcriptional enhancer elements; we have shown that some of these interact with the GREM1 promoter and are capable of driving gene expression in vitro. Increased GREM1 expression is predicted to lead to reduced bone morphogenetic protein pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. The pathogenic mechanism in HMPS is extremely unusual in Mendelian cancer syndromes and highlights ectopic gene expression as a mechanism of tumorigenesis.