Variants in Hdac9 Intronic Enhancer as Candidates for Skin Tumor Susceptibility Locus Tyler E. Siekmann1 and Amanda E. Toland1* Biomedical Science Undergraduate Program1, The Ohio State University Department of Molecular Virology, Immunology and Medical Genetics2 Abstract Enhancer Activity Demonstrated in Hdac9 Non-melanoma skin cancers (NMSC) are the most common forms of cancer in the world accounting for nearly half of all cancer diagnoses. Rates of NMSC are on the rise with an over 300% increase in diagnosis of these cancers in the last 20 years. While environmental risk factors for skin cancers are well understood, little is known about genetic risk factors for these cancers. Mouse linkage studies have identified several loci housing skin cancer susceptibility genes. Human tumors show evidence of preferential allelic imbalance for polymorphisms in Hdac9, a gene mapping to one of the linkage regions, Skts5. One intron in the Hdac9 gene between exons 8 and 9 contains an enhancer for Twist1, affecting limb development and phenotypes in the skin. Twist1 is a known regulator of skin differentiation and has a documented role in cancer. The hypothesis of this study is that this enhancer locus plays a role in the differential risk for NMSC between the cancer susceptible NIH/Ola and cancer resistant Spretus/EiJ mice. Sequence analyses identified several polymorphisms between these strains in this intron which are predicted by in silico methods to disrupt transcription factor binding. To investigate in vitro effects of these variants, intron fragments from both NIH/Ola and Spret/EiJ murine DNA were cloned into an enhancer reporting PGL3 vector and transfected into both normal keratinocyte C5N and squamous cell carcinoma A5 cells. Luciferase assay and real-time PCR data suggest these variants are responsible for changes in gene expression, specifically in the Twist1 gene. Chromatin Immunoprecipitation studies are being performed to test whether transcription factors, Oct1 and Gata3 that are predicted to differentially bind the NIH/Ola and Spret/EiJ enhancer , are involved in the differential Twist1 expression. This project has the potential implication of discovering the role a specific gene locus, Skts5, plays in NMSC risk. A We hypothesize that variants in the Hdac9 intron 8 Enhancer Locus play a role in the differential risk for Non-melanoma Skin Cancers observed between the cancer susceptible NIH/Ola and the cancer resistant Spretus mouse strains. Identification of Polymorphisms Between NIH & Spretus Murine DNA at Hdac9 Intron • Custom primers were designed to break up Hdac9 intron 8 into smaller fragments capable of being sequenced • In total, 43 polymorphisms were found between cancer susceptible NIH/Ola and cancer resistant Spretus mice DNA A Spret B EXON 1 2 3 4 5 6 7 8 Ch. 12: 34902877 9 10 34917095 Intron 8-9 Intron Fragment # Broken Up By Custom Primers NIH 1 2 3 4 5 6 7 8 9 *Figure not to scale Figure 1: A) Examples of sequence variants within Hdac9 intron 8 between NIH & Spret B) Map of Hdac9 gene with Intron 8 magnified. Custom primer inserts shown visually. Quantification of White Pixels Quantification of White Pixels 25000 20000 15000 10000 Figure 2: Cells transfected with pGL3 + NIH Inserts 1 & 2 show significantly higher Luciferin expression than those transfected with pGL3 + Spret Inserts 1 & 2 in both C5N normal keratinocyte and A5 SCC cell lines. Insert 1: p<0.01**, Insert 2: p<0.05* • Enhancer activity exists in the Insert 1 & 2 region of Hdac9 Intron 8 • This enhancer activity is unique to the NIH mouse strain, not found in Spretus • Enhancer activity does not extend to later portions of the intron (i.e. Insert 7) Hdac9 Enhancer Affects Twist1 Expression Average % Twist1 Expression Relative to Hprt B 20 15 10 TWIST1 5 HDAC9 Spretus 1 Samples Figure 3: A) Real-Time PCR Data indicating cells transfected with pGL3+NIH Insert 1 display a near 2 fold increase in Twist1 expression compared to pGL3 control and pGL3+Spretus Insert 1 in A5 cells (p<0.05). B) Map of proposed genetic interaction • Twist1 identified as a target gene of the Hdac9 Intron 8 enhancer activity • Similar study looking at Hdac9 did not see significant differences in expression Transcription Factor Binding Predictions Oct1 Gata3 CdxA Lyf-1 Gfi-1 Nkx-2 AP-1 20000 15000 10000 0 NIH Insert 1 Region 2 Spret Insert 1 Region 2 pGL3 Control NIH Insert 1 Region 2 Spret Insert 1 Region 2 B Figure 4: A) Quantification of ChIP results for Oct1 and Gata3 Transcription Factors (TF). B) Gel images of ChIP results for the respective TFs (C5N cells) • • • • Oct1 shown to preferentially bind NIH Insert 1 in comparison to Spretus Insert 1 Gata3 shown to preferentially bind Spretus Insert 1 in comparison to NIH Insert 1 Both in silico predictions validated through in vitro ChIP studies Implications for these factors in the functionality of the Hdac9 enhancer Conclusions and Impact • An intronic enhancer in the Hdac9 gene was identified in mice • This intronic enhancer was shown to be present in cancer susceptible NIH/Ola mice but not in cancer resistant Spretus mice • A relation between this enhancer and Twist1 upregulation was demonstrated • Two transcription factors, Oct1 and Gata3, were shown to differentially bind one of the two mice DNA strains, suggesting a possible mechanism for SCC risk at Skts5 • One of the first studies analyzing the role of intronic enhancers in skin cancer risk 25 NIH 1 Samples 25000 Mouse Genetic Locus of Hdac9 & Twist1 30 PGL3 Control Samples 30000 5000 pGL3 Control Transcription Factor HDAC9 35000 0 Mock Samples Determine if variants exist between NIH and Spretus DNA within Hdac9 intron 8. Identify and characterize any enhancer sites in the murine Hdac9 intron 8 sequence Determine which gene(s) are targets of the enhancer(s) and demonstrate via qPCR Identify specific transcription factor/DNA binding interactions potentially associated with enhancer activity using Chromatin Immunoprecipitation Assays 40000 5000 0 The Specific Aims of this project include the following: ChIP: Gata3 Binding to Hdac9 30000 % Twist1 Expression to Hprt Hypothesis and Specific Aims ChIP: Oct1 Binding to Hdac9 35000 A 1. 2. 3. 4. Oct1 and Gata3 Transcription Factors Differentially Bind NIH & Spretus in vitro DNA Binding Site Polymorphism Binding Strain Significance Score ATATACACT AATCACG ATATAG TGGGAT ATAGTTGTGAT TCAAGTG GTGATTAA C/G C/T G/C A/G A/G C/A A/G Spretus NIH NIH Spretus Spretus Spretus NIH 89.7 85.9 85.0 85.7 85.9 89.9 86.4 Table 1: In silico predictions of transcription factors that will differentially bind NIH and Spretus DNA at the Hdac9 Enhancer locus. Databases utilized include TFsearch, DBD, PROMO(Transfac) and TFSiteScan. Cutoff score of 85.0 used for significance. Acknowledgements Special thanks should be given to the members of the Toland Laboratory, the Ohio State University Biomedical Science Major and the Pelotonia Undergraduate Fellowship Program (1) Fleming J, Toland A. E. “Allele-specific imbalance mapping identifies HDAC9 and IFRD1 as candidate susceptibility genes for cutaneous squamous cell carcinoma”. Journal of Investigative Dermatology, 2011. (2) Ahituv N. “Dual function of DNA sequences: Coding exons function as enhancers of nearby genes”. Abstract 13, American Society of Human Genetics Annual Meeting, Montreal, 2011. (3) VanderMeer J, Ahituv N. “Cis-Regulatory Mutations Are a Genetic Cause of Human Limb Malformations”. Developmental Dynamics, 2011; 240:920-930. This work was supported by the Pelotonia Fellowship Program. Any opinions, findings, and conclusions expressed in this material are those of the author’s and do not necessarily reflect those of the Pelotonia Fellowship Program.
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