1: J Clin Invest. 2004 Oct;114(8):1146-57. Epigenetic regulation of 11 beta-hydroxysteroid dehydrogenase type 2 expression. Alikhani-Koopaei R, Fouladkou F, Frey FJ, Frey BM. Department of Nephrology and Hypertension, University Hospital of Berne, Berne UNK 3010, Switzerland. The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11 beta-hydroxyglucocorticoids. Variable activity of 11 beta HSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11 beta HSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2'-deoxycytidine and procainamide enhanced the transcription and activity of the 11 beta HSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter-luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG-binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension. PMID: 15489962 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Exp Mol Med. 2003 Oct 31;35(5):393-402. Genomic organization and expression of parkin in Drosophila melanogaster. Bae YJ, Park KS, Kang SJ. Department of Biological Science, College of Natural Science, Ewha Womans University, Seoul 120-750, Korea. We report here the isolation, characterization on genomic structure and expression of the D. melanogaster homolog of human parkin. The 2,122 bp parkin gene sequence contains six exons that form a 1,449 bp transcript encoding a protein of 482 amino acids. 151 bp of 5' and 112 bp of 3' untranslated regions were identified by a combination of 5'-RACE/primer extension and 3'-RACE, respectively. The 5' UTR contains three transcription initiation sites. Neither a classical TATA nor a CAAT box was found in the putative promoter sequence. However, binding sites for AhR-Arnt, AP4, NF1 and GATA transcription factors were identified. Transient transfection analysis of the 5' UTR confirmed its promoter activity in HEK 293 cells and SH-SY5Y neuronal cells using a dual luciferase reporting system. The amino acid sequence of D. melanogaster Parkin exhibits 42%, 43% and 43% identity to that of human, mouse and rat, respectively, representing a 54 kDa protein band via western blot analysis. It shows a high degree of conservation in the Ubiquitin-like domain at the N-terminus (34%), the In-Between RING finger domains (IBR, 65-69%), and the RING finger domains at the C-terminus (56-57%). The expression pattern of D. melanogaster parkin varies during the developmental stages, with the highest expression in the adult stage as measured by competitive RT-PCR. From immunostainings of the embryo, D. melanogaster parkin was expressed slightly higher in the central nervous system (brain and nerve cord) during the late embryonic stage. PMID: 14646593 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Adv Exp Med Biol. 1999;463:159-64. Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids. Lindahl R, Xiao GH, Falkner KC, Prough RA. Department of Biochemistry, University of South Dakota School of Medicine, Vermillion 57069, USA. The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene. PMID: 10352681 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------