Browsing by Author "Jairam, Sowmya"

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    An enhancer- blocking element regulates the cell-specific expression of alcohol dehydrogenase 7
    (Elsevier, 2014-09-01) Jairam, Sowmya; Edenberg, Howard J.; Department of Biochemistry and Molecular Biology, IU School of Medicine
    The class IV alcohol dehydrogenase gene ADH7 encodes an enzyme that is involved in ethanol and retinol metabolism. ADH7 is expressed mainly in the upper gastrointestinal tract and not in the liver, the major site of expression of the other closely related ADHs. We identified an intergenic sequence (iA1C), located between ADH7 and ADH1C, that has enhancer-blocking activity in liver-derived HepG2 cells that do not express their endogenous ADH7. This enhancer blocking function was cell- and position-dependent, with no activity seen in CP-A esophageal cells that express ADH7 endogenously. iA1C function was not specific to the ADH enhancers; it had a similar cell-specific effect on the SV40 enhancer. The CCCTC-binding factor (CTCF), an insulator binding protein, bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of ADH enhancers and thereby contributes to the cell specificity of ADH7 expression.
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    Single-nucleotide polymorphisms interact to affect ADH7 transcription
    (Wiley Blackwell (Blackwell Publishing), 2014-04) Jairam, Sowmya; Edenberg, Howard J.; Department of Biochemistry and Molecular Biology, IU School of Medicine
    BACKGROUND: The class IV alcohol dehydrogenase (ADH7, μ-ADH, σ-ADH) is important in the metabolism of ethanol and retinol. ADH7 is the only ADH not expressed in liver, instead being expressed mainly in the upper gastrointestinal tract. Genome-wide studies have identified significant associations between single-nucleotide polymorphisms in ADH7 and alcoholism and cancer, but the causative variants have not been identified. METHODS: In vitro studies of gene expression by transient transfection into cell lines that express endogenous ADH7 (CP-A cells) and that do not (HepG2 cells). RESULTS: We have identified transcriptional regulatory elements of ADH7 and observed differences in the effects of variants on gene expression in CP-A cells and HepG2 cells. Two haplotypes of the proximal promoter that differ in a single nucleotide at rs2851028, A7P-G and A7P-A, have different transcriptional activities. There is an interaction between variants farther upstream and these proximal variants: Upstream regulatory sequences generally showed a greater increase or smaller reduction in activity when combined with the A7P-A promoter than with the A7P-G promoter. A sequence located 12.5-kb upstream (7P10) can function as an enhancer. In CP-A cells, both haplotypes of 7P10 increased A7P-A activity by 2.5-fold while having only 1.2-fold effect on A7P-G. In HepG2 cells, the 7P10-TTT haplotype had no effect on the A7P-A promoter but decreased A7P-G promoter activity by 50%, whereas the CTT haplotype increased A7P-A activity by 50%, but had no effect on A7P-G. CONCLUSIONS: These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context and should be considered in interpretation of the association of variants with alcoholism and cancer.
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    Transcription regulation of the class II alcohol dehydrogenase 7 (ADH7)
    (2014-05) Jairam, Sowmya; Edenberg, Howard J.; Herring, B. Paul; Skalnik, David Gordon; Wek, Ronald C.
    The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.