The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5’ gene region which we sought to investigate more fully.We evaluated the 5‘ end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5’ untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5’ untranslated region is affected by the (GT)n microsatellite.This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.
Copy number variations (CNVs) were found to contribute massively to the variability of genomes. One of the best studied CNV region is the b-defensin cluster (DEFB) on 8p23.1. Individual DEFFB copy numbers (CNs) between 2 and 12 were found, whereas low CNs predispose for Crohn's disease. A further level of complexity is represented by sequence variations between copies (multisite variations, MSVs). To address the relation of DEFB CN and MSV to the expression of b-defensin genes, we analyzed DEFB4 expression in B-lymphoblastoid cell lines (LCLs) and primary keratinocytes (normal human epidermal keratinocyte, NHEK) before and after stimulation with lipopolysaccharide, tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g). Moreover, we quantified one DEFB4 MSV in DNA and mRNA as a marker for variant-specific expression (VSE) and resequenced a region of B2 kb upstream of DEFB4 in LCLs. We found a strong correlation of DEFB CN and DEFB4 expression in 16 LCLs, although several LCLs with very different CNs exhibit similar expression levels. Quantification of the MSV revealed VSE with consistently lower expression of one variant. Costimulation of NHEKs with TNF-a/IFN-g leads to a synergistic increase in total DEFB4 expression and suppresses VSE. Analysis of the DEFB4 promoter region showed remarkably high density of sequence variabilities (B1 MSV/41 bp).
BackgroundAcid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.Methodology/Principal FindingsWe identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.Conclusions/SignificanceThese findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.
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