Entire Promoter Region Research Articles

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

Novel Mutations Identified in the Chinese Han Population with Keratoconus by Next-Generation Sequencing.

Aim To identify novel mutations in keratoconus (KC) susceptibility genes in the Chinese Han population. Methods A total of fifty-two patients with primary KC were recruited. Blood samples were collected, and genomic DNA was isolated from peripheral blood leukocytes. The entire coding region, intron-exon junctions, and promoter regions of sixteen known KC susceptibility genes were screened with next-generation sequencing technology. All identified variants were further confirmed using the Sanger sequencing technology. The Sorting Intolerant from Tolerant (SIFT), MutationTaster, and PolyPhen 2 programs were used to predict the effect of amino acid substitution on protein. Results After removing twelve known SNPs (single nucleotide polymorphisms) and three variants predicted to be harmless, nine novel mutations were identified in eight of the fifty-two patients, including c.455C > T:p.P152L in FNDC3B; c.3636_3637del:p.R1212fs in COL4A4; c.5015G > T:p.R1672L, c.3798dupA:p.P1267fs, and c.28G > A:p.A10T in MPDZ; c.1940C > T:p.P647L in DOCK9; c.127_128insGGC:p.Q43delinsRQ in POLG; c.3019G > A:p.V1007I in IPO5; and c.624 + 7− > A in TGFBI. All nine mutations in the patients with KC were heterozygote. Conclusion This study enlarged the gene profile of KC and should be further confirmed by well-powered, genome-wide association studies (GWAS) of Han Chinese patients.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression.

The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation.

SNP Diversity in CD14 Gene Promoter Suggests Adaptation Footprints in Trypanosome Tolerant N'Dama (Bos taurus) but not in Susceptible White Fulani (Bos indicus) Cattle.

Immune response to infections has been shown to be mediated by genetic diversity in pattern recognition receptors, leading to disease tolerance or susceptibility. We elucidated naturally occurring variations within the bovine CD14 gene promoter in trypanosome-tolerant (N’Dama) and susceptible (White Fulani) cattle, with genomic and computational approaches. Blood samples were collected from White Fulani and N’Dama cattle, genomic DNA extracted and the entire promoter region of the CD14 gene amplified by PCR. We sequenced this region and performed in silico computation to identify SNP variants, transcription factor binding sites, as well as micro RNAs in the region. CD14 promoter sequences were compared with the reference bovine genome from the Ensembl database to identify various SNPs. Furthermore, we validated three selected N’Dama specific SNPs using custom Taqman SNP genotyping assay for genetic diversity. In all, we identified a total of 54 and 41 SNPs at the CD14 promoter for N’Dama and White Fulani respectively, including 13 unique SNPs present in N’Dama only. The significantly higher SNP density at the CD14 gene promoter region in N’Dama may be responsible for disease tolerance, possibly an evolutionary adaptation. Our genotype analysis of the three loci selected for validation show that mutant alleles (A/A, C/C, and A/A) were adaptation profiles within disease tolerant N’Dama. A similar observation was made for our haplotype analysis revealing that haplotypes H1 (ACA) and H2 (ACG) were significant combinations within the population. The SNP effect prediction revealed 101 and 89 new transcription factor binding sites in N’Dama and White Fulani, respectively. We conclude that disease tolerant N’Dama possessing higher SNP density at the CD14 gene promoter and the preponderance of mutant alleles potentially confirms the significance of this promoter in immune response, which is lacking in susceptible White Fulani. We, therefore, recommend further in vitro and in vivo study of this observation in infected animals, as the next step for understanding genetic diversity relating to varying disease phenotypes in both breeds.

The high-pathogenicity island (HPI) promotes flagellum-mediated motility in extraintestinal pathogenic Escherichia coli.

The key of success of extraintestinal pathogenic Escherichia coli (ExPEC) to colonize niches outside the intestinal tract and to establish infection is the coordinated action of numerous virulence and fitness factors. The so-called high-pathogenicity island (HPI), responsible for synthesis, secretion and uptake of the siderophore yersiniabactin, proved to be an important virulence determinant. In this study we investigated the interaction of the flagellum-mediated motility and the HPI. The impairment of yersiniabactin production by deletion of irp2 or ybtA affected significantly motility. The gain of yersiniabactin production improved motility in both pathogenic and non-pathogenic E. coli strains. The loss of flagella expression had no adverse effect on the HPI. Strikingly, external iron abundance was not able to suppress activation of the HPI during motility. The HPI activity of swarming bacteria was comparable to iron deplete conditions, and could even be maximized by supplementing excessive iron. This fact is the first description of a regulatory mechanism, which does not follow the known hierarchical regulation of siderophore systems. Transcriptional reporter fusions of the ybtA promoter demonstrated that the entire promoter region with all YbtA binding sites is necessary for complete induction in both HPI-positive and HPI-negative strains. Altogether, these results suggest that the HPI is part of a complex regulatory network, which orchestrates various virulence mechanisms to optimize the overall fitness of ExPEC.

Comparison of genomic alterations in bladder urothelial tumors with and without telomerase reverse transcriptase promoter mutation using a next-generation sequencing assay.

310 Background: Telomerase reverse transcriptase (TERT) is the most frequently altered gene in urothelial cancer (UC), detected across all grades and stages of disease. We sought to characterize TERT alterations within a prospective cohort of UC treated at our institute and compare the frequency of genomic alterations in TERT promoter mutant vs wild-type UC specimens. Methods: Patients diagnosed with bladder urothelial tumors were enrolled onto an institutional review board approved prospective sequencing protocol. Tumor and matched germline DNA were analyzed for somatic point mutations, truncations, copy number alterations, and insertions/deletions using the MSK-IMPACT NGS assay that detects alterations in all exons and select introns of 410 oncogenes and tumor suppressor genes as well as the entire TERT promoter region. Results: 329 UC were sequenced of which 236 (71.7%) harbored TERT mutations, the majority being promoter region hotspots (chr5: 1295228 G > A [81%] and chr5:1295250 G > A [16%]). Patients with TERT promoter mutations were significantly older than those without (69.01±10.70 years vs. 65.44±11.78 years, p = 0.0317). UC with TERT promoter mutations had significantly higher mutation count [median 10 (range: 2-76) vs median 5 (range: 0-119)] as well as copy number alterations [median 0.11 (range: 0-0.68) vs median 0.047 (range: 0-0.65)]. Between UC with and without TERT promoter mutations, there was a very significant difference in mutation frequencies of ARID1A(33% vs 11%), PIK3CA(28% vs 13%), FGFR3(32% vs 16%), CREBBP(19% vs 4%), CDKN1A(17% vs 2%), ERBB2(26% vs 9%), ERCC2(15% vs 3%), TSC1(11% vs 0%), MT2C(17% vs 9%) (all with p < 0.005). Conclusions: TERT is the most frequently altered gene in bladder cancer with the majority of TERT alterations comprised of two hotspot mutations. UC with TERT promoter mutations tends to occur in older patients and is associated with overall higher mutation count and copy number alterations. A number of genes are differentially mutated in UC with and without TERT promoter mutations and may suggest a link between TERT promoter mutations and distinct mutations profiles in UC.

Novel SNPs and InDels discovered in two promoter regions of porcine pregnancy-associated glycoprotein 2-like subfamily (pPAG2-Ls) in crossbreed pigs.

This is a pioneer study of single nucleotide polymorphisms (SNPs) within the entire promoter region (1204 bp) of the dominant pPAG2-L subfamily in the pig. The pPAG2-L subfamily was sequenced/examined using genomic deoxyribonucleic acid (gDNA) templates of crossbreed pigs (Landrace x Large White), and compared to two bacterial artificial chromosome (BAC) clones containing gDNA of the Duroc breed (as the positive controls). Our analysis of the pPAG2-L promoter identified 31 SNPs and one InDel mutation in crossbreed pigs. Among 42 SNPs identified in two BAC clones, 24 SNPs had not been previously detected in crossbreed pigs. The sequence alignment of pPAG2-L promoter, performed with Lasagne-Search 2.0, Cluster Bluster and MatInspector software, revealed a total of 28 transcription factor binding sites (TFBS) and 10 TFBS (AP-1, CCAAT, CHOP:C, FOXP1, LSF, MRF-2, Myc, NF1, NF-Y, TGIF) within SNPs in the core sequences. It was noted that TFBS (NF1) was found to be unique to the pPAG2 promoter sequence containing SNPs: g.-1100G>A(R), g.-1101T>C(Y), represented by GA and TC genotypes (p x = 0.12). Our broad-based novel database thus provides an SNP PAG2-L pattern for modern genotyping of female and male progenitors. This is required for further studies of various potential correlations between guiding SNP genotypes of the pPAG2-L subfamily in the sows of many breeds, in which the most economically important reproductive traits are properly documented on each farm.

B15 - Polymorphism of CDH1 Promoter Is a Predictor of Clinical Outcome in Patients with Metastatic Gastric Cancer Treated with chemotherapy

Background: CDH is a Ca2+-dependent cell adhesion molecule with tumor metastasis suppressor activity. Mutations of CDH1 and its E-cadherin product have been associated with the origin, development, invasion, metastasis, and prognosis of gastric cancer (GC). The present study analyzed CDH1 gene polymorphisms and their impacts on chemotherapeutic event free response (EFS) and survival (OS) in patients with metastatic gastric cancer (MGC) treated with DOC2 (Docetaxel, Oxaliplatin) regimen. The original chemotherapeutic program DOC was developed at the CRO - Cancer Center in Aviano. The phase I study determined the Maximum Tolerated Dose (MTD) and, as per protocol, indicated the level just below the MTD as appropriate for a phase II study. Patients and methods: This is a multi-centre, phase II single arm study of DOC combination in patients with previously untreated surgically unresectable or metastatic gastric cancer. Eligible subjects will be enrolled to receive first-line combination therapy consisting of DOC2 treatment. The foreseen schedule consists of: Docetaxel 30 mg/m2, Oxaliplatin 70 mg/m2, both i.v., on days 1 and 8, associated to Capecitabine 500 mg/m2, p.o., bid, on day 2 to 15, on a three week basis. The study has been designed to determine the activity and safety of the DOC regimen in previously untreated patients. Each patient will be treated for a maximum of 6 cycles until disease progression, unacceptable toxicity, or patient refusal, whichever occur first. However, Capecitabine 1000 mg/m2/die tablets p.o. alone is allowed in patients without tumor progression at the end of chemotherapy (maintenance treatment) for further 4-6 cycles. Up to now the study enrolled 29 out 71 foreseen MGC patients treated with first-line DOC2 chemotherapy with a mean follow-up of 1.2 years (range 0.2 to 3.2). Polymorphisms of the entire CDH1 exons and promoter regions were determined. Results: Among the 37 regions we analyzed, polymorphism in the promoter (P1) was significantly associated with EFS (HR 4.8, 95%CI 1.8-12.7, p = 0.004) and showed an increasing trend of OS (HR 2.8, 95%CI 0.9-8.7). Conclusions: Our result suggest promoter region of CDH1 might be independent predictor of chemotherapeutic clinical outcome.

A polymorphic minisatellite region of BORIS regulates gene expression and its rare variants correlate with lung cancer susceptibility.

Aberrant expression of BORIS/CTCFL (Brother of the Regulator of Imprinted Sites/CTCF-like protein) is reported in different malignancies. In this study, we characterized the entire promoter region of BORIS/CTCFL, including the CpG islands, to assess the relationship between BORIS expression and lung cancer. To simplify the construction of luciferase reporter cassettes with various-sized portions of the upstream region, genomic copies of BORIS were isolated using TAR cloning technology. We analyzed three promoter blocks: the GATA/CCAAT box, the CpG islands and the minisatellite region BORIS-MS2. Polymorphic minisatellite sequences were isolated from genomic DNA prepared from the blood of controls and cases. Of the three promoter blocks, the GATA/CCAAT box was determined to be a critical element of the core promoter, while the CpG islands and the BORIS-MS2 minisatellite region were found to act as regulators. Interestingly, the polymorphic minisatellite region BORIS-MS2 was identified as a negative regulator that repressed the expression levels of luciferase reporter cassettes less effectively in cancer cells compared with normal cells. We also examined the association between the size of BORIS-MS2 and lung cancer in a case–control study with 590 controls and 206 lung cancer cases. Rare alleles of BORIS-MS2 were associated with a statistically significantly increased risk of lung cancer (odds ratio, 2.04; 95% confidence interval, 1.02–4.08; and P=0.039). To conclude, our data provide information on the organization of the BORIS promoter region and gene regulation in normal and cancer cells. In addition, we propose that specific alleles of the BORIS-MS2 region could be used to identify the risk for lung cancer.

9B.02

Objective: We previously showed that a blunted expression of the Twik-related Acid-Sensitive K+ channel 2 (TASK-2) is a common feature of Aldosterone Producing Adenoma (APA). Thus, we aimed at investigating the presence of mutations in the promoter region of the TASK-2 gene (KCNK5) in APA. Design and method: We sequenced the entire TASK-2 promoter region of 88 APA and 98 primary hypertensive patients (controls). We next tested the in vitro effects of the most frequently detected mutation by fusing the mutated and wild type TASK-2 promoter region to the luciferase coding sequence and transfecting the reporter vectors in H295R cells. Results: We detected only one mutation (C999T) in one of 98 primary hypertensive patients. Seven novel mutations were found in 16% of APA: the C999T variant was found in 6% of APA, the G595A in 3.5% of APA, the G36A in 2% of APAs, while the Gins466, G263C, C1247T and G1140T mutations in 1% of APA. None was germline. By site-direct mutagenesis we demonstrated that the C999T mutation significantly decreased the TASK-2 transcription activity and luciferase signal of the reporter vector in transfected H295R cells (fold change of normalized luciferase signal: 0.55 ± 0.22, p Conclusions: Thus, we demonstrated that 16% of APA have seven recurrent mutations in the promoter region of TASK-2 gene. One of these TASK-2 genetic variants blunts the transcription of the TASK-2 in human adrenal cells, thus suggesting a possible molecular mechanism contributing to the autonomous aldosterone secretion typical of APA.

Prognostic prediction of glioblastoma by quantitative assessment of the methylation status of the entire MGMT promoter region.

BackgroundO6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is reported to be a prognostic and predictive factor of alkylating chemotherapy for glioblastoma patients. Methylation specific PCR (MSP) has been most commonly used when the methylation status of MGMT is assessed. However, technical obstacles have hampered the implementation of MSP-based diagnostic tests. We quantitatively analyzed the methylation status of the entire MGMT promoter region and applied this information for prognostic prediction using sequencing technology.MethodsBetween 1998 and 2012, the genomic DNA of 85 tumor samples from newly diagnosed glioblastoma patients was subjected to bisulfite treatment and subdivided into a training set, consisting of fifty-three samples, and a test set, consisting of thirty-two samples. The training set was analyzed by deep Sanger sequencing with a sequencing coverage of up to 96 clones per sample. This analysis quantitatively revealed the degree of methylation of each cytidine phosphate guanosine (CpG) site. Based on these data, we constructed a prognostic prediction system for glioblastoma patients using a supervised learning method. We then validated this prediction system by deep sequencing with a next-generation sequencer using a test set of 32 samples.ResultsThe methylation status of the MGMT promoter was correlated with progression-free survival (PFS) in our patient population in the training set. The degree of correlation differed among the CpG sites. Using the data from the top twenty CpG sites, we constructed a prediction system for overall survival (OS) and PFS. The system successfully classified patients into good and poor prognosis groups in both the training set (OS, p = 0.0381; PFS, p = 0.00122) and the test set (OS, p = 0.0476; PFS, p = 0.0376). Conventional MSP could not predict the prognosis in either of our sets. (training set: OS; p = 0.993 PFS; p = 0.113, test set: OS; p = 0.326 PFS; p = 0.342).ConclusionsThe prognostic ability of our prediction system using sequencing data was better than that of methylation-specific PCR (MSP). Advances in sequencing technologies will make this approach a plausible option for diagnoses based on MGMT promotor methylation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-641) contains supplementary material, which is available to authorized users.

Abstract 3821: Elucidating the role of PBRM1 in SNF5 regulated gene expression in malignant rhadboid tumors.

Abstract Malignant Rhabdoid Tumors (MRTs), a pediatric renal cancer, lack SNF5, a subunit of the SWI/SNF chromatin remodeling complex, a regulator of nucleosome positioning and gene expression. Recent studies showed that PBRM1, another subunit of SWI/SNF, was mutated in adult clear cell renal carcinoma (ccRCC). Intriguingly, both SNF5 and PBRM1 regulate the cell cycle through control of expression of p21WAF1/CIP1. We hypothesized that SWI/SNF complex mutations provide a link between adult and pediatric renal cancers. To test this notion, we re-expressed SNF5 in MRT cell lines and followed PBRM1 and p21 expression and cell growth. We also examined SWI/SNF complex composition and recruitment to the p21 promoter. Finally, we investigated PBRM1’s role in SNF5-induced G1 cell cycle arrest using PBRM1-deficient MRT cell lines. We utilized adenoviral vectors to express human SNF5 (hSNF5) in several different MRT cell lines. The expression of hSNF5 and other complex members was quantified through immunoblotting and QT-PCR. Immunoprecipitation (IP) was utilized to determine the composition of the SWI/SNF complex. Cell cycle analysis was conducted using flow cytometry with BrdU and PI staining. PBRM1 knockdown cell lines were generated using lentiviral-RNAis (Open Biosystems). Reexpression of SNF5 in MRT cell lines led to increased PBRM1 protein levels without concomitant increases in mRNA levels. Immunoprecipitation experiments showed that SNF5 re-expression caused PBRM1 levels in the complex to increase, as compared to the BRG1 ATPase levels that remained constant after SNF5 reexpression. These results were consistent across 4 MRT cell lines examined. Interestingly, levels of other complex proteins, such as BAF155, also increased in a similar fashion to PBRM1. After reexpression of SNF5, we also observed an increase of both SNF5 and PBRM1 at the p21 promoter, with a peak at the transcription start site. In contrast, other complex members showed only a modest increase across the entire promoter region with no apparent peak of binding. To further investigate PBRM1’s role in SNF5-induced G1 cell cycle arrest, we generated PBRM1-deficient MRT cell lines using RNAi technology. We are currently testing these knockdown cell lines for cell cycle arrest and p21 induction after hSNF5 reexpression. Our data show that SNF5 expression increases PBRM1 protein levels, presumably through stabilization within the SWI/SNF complex, suggesting a high degree of interplay between PBRM1 and SNF5 during chromatin remodeling activity. Therefore, identifying genes regulated by both proteins will prove critical in further understanding their contribution to MRT development. Ultimately, our studies will identify key signaling pathways that contribute to the initiation and/or progression of renal cancers including MRTs and ccRCCs. Citation Format: Darmood Wei, Bernard E. Weissman, Yasumichi Kuwahara. Elucidating the role of PBRM1 in SNF5 regulated gene expression in malignant rhadboid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3821. doi:10.1158/1538-7445.AM2013-3821

Expression of PRPF31 and TFPT: regulation in health and retinal disease

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Transcriptional regulation of the human mitochondrial peptide deformylase (PDF)

The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8’s coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner.

Determination of the DNA Binding Mode of the Transcriptional Repressor ICER

Inducible cAMP Early Repressor (ICER) is a leucine zipper protein, a product of the cAMP Responsive Element Modulator (CREM) gene. ICER functions as a transcriptional repressor and its expression is abnormal in certain types of cancer where ICER acts as a tumor repressor. ICER binds to cAMP Responsive Elements (CRE's) found in the promoter sequences of genes involved in cellular growth. It also binds to the four CRE sites on its own promoter, thereby regulating its own expression. These four sites located on the CREM gene promoter are designated CARE-1 through CARE-4, and each one consists of 8 to 9 bases. On the basis of the presence of four CRE sites, we hypothesize that ICER may autoregulate its expression by cooperative binding to its own promoter. To test this hypothesis, we are performing titrations of purified ICER with double stranded DNA that is labeled with a fluorophore-quencher pair and monitor the binding by measuring the change in fluorescence due to an increase in resonance energy transfer upon a decrease in fluorophore-quencher distance. The dsDNA contains one or more of the CARE sites. We are determining the dissociation constant of ICER for each of the four CARE sites as well as examine the potential for cooperative binding between multiple CARE sites. Experiments on dsDNA (35 bp) with CARE-1 and/or CARE-2 have each yielded a dissociation constants of kd = 0.16 ± 0.02 μM, and no obvious evidence for cooperative binding between CARE-1 and CARE-2 has yet been observed. Analysis of dsDNA with CARE-3 and CARE-4 as well as for the entire promoter region with all four CARE sites will be presented and a model for binding of ICER to its own promoter will be discussed.

Abstract LB-84: DNA methylation centers in CpG-island promoters are context-specific

Abstract Repression of many TSGs (tumor suppressor genes) in cancer is mediated by aberrantly increased DNA methylation levels of CpG islands at promoters. Evidence from plants and mammalian cells indicate that sequences within or around target sites could influence DNA methylation patterns. However, it is still unknown whether aberrant methylation in cancer starts randomly or is triggered by specific DNA sequences. Here, we test the hypothesis that cisacting elements trigger de novo CpG-island promoter methylation. We first examined methylation patterns of the RIL promoter region (from −722 to +630 from transcription start) in several cell types and tissues. RIL is a candidate tumor suppressor gene that is frequently hypermethylated in cancer. Bisulfite-sequencing results identified 11 CpG sites upstream and 17 CpG sites downstream that are densely methylated both in normal blood (average methylation density, upstream 82.2± 11.6% and downstream 73.6± 17.7%) and colon tissues (upstream 80.5±14.6% and downstream 76.2±12.1%), compared with those within the CpG island (17.6± 10.0% in blood and 20.2±15.0% in colon tissues). This sharp transition of methylation surrounding the CpG island has also been seen in some genes such as p16, VHL and CDH1. We hypothesized that these highly methylated CpG sites in normal tissues serve as methylation centers that attract DNMT binding and trigger de-novo DNA methylation. To test this hypothesis while controlling for insertion site effects, we established a system using Flp/FRT to construct single integration sites in a host cell line (HCT116 colon cancer cell line), so that exogenous fragments from CpG-island promoters can be introduced into the same genomic locus or chromatin micro-environment. The entire RIL promoter region (−713 to +664, 96 CpG sites), or isolated fragments containing the hypermethylated upstream (−713 to −378, 11 CpG sites) and downstream regions (+214 to +664, 20 CpG sites), and the central part (−406 to +237, 66 CpG sites) were integrated (separately) into a single genomic locus at 3q13.31, corresponding to the intron of an expressed gene. Cells collected at 1, 2 and 3 months after transfection were examined by bisulfite-sequencing. No significant de-novo methylation of the transfected constructs was observed. We next tested a genomic region highly methylated in all tissues examined except germ cells, the insulin-6 promoter, as well as 3 motifs in this promoter computationally associated with DNA methylation, but both constructs remained completely methylation free after transfection. Thus, multiple sequences associated with very high levels of DNA methylation in normal and neoplastic tissues can be completely methylation free when inserted into a different chromatin context. These results have important implications for the pathogenesis of aberrant methylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-84.

High resolution map of Caenorhabditis elegans gap junction proteins

The innexin family of gap junction proteins has 25 members in Caenorhabditis elegans. Here, we describe the first high-resolution expression map of all members through analysis of live worms transformed with green fluorescent protein under the control of entire promoter regions. Our analyses show that innexins have dynamic expression patterns throughout development and are found in virtually all cell types and tissues. Complex tissues, such as the pharynx, intestine, gonad, as well as scaffolding tissues and guidepost cells express a variety of innexins in overlapping or complementary patterns, suggesting they may form heteromeric and heterotypic channels. Innexin expression occurs in several types of cells that are not known to form gap junctions as well as in a pair of migrating cells, suggesting they may have hemichannel function. Therefore, innexins likely play roles in almost all body functions, including embryonic development, cell fate determination, oogenesis, egg laying, pharyngeal pumping, excretion, and locomotion.

Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability

It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.