Gene structure and promoter for Adh3 encoding mouse class IV alcohol dehydrogenase (retinol dehydrogenase).

Class IV alcohol dehydrogenase (ADH) has been shown to function in vitro as a retinol dehydrogenase catalyzing the synthesis of retinoic acid, a pleiotropic gene regulator. To enable genetic studies on the function of this enzyme and regulation of its gene, we have screened a genomic library and isolated the mouse class IV ADH gene (Adh3). The complete mouse class IV ADH coding region was found in nine exons spanning a 14-kb region. Primer extension analysis was used to map the transcription initiation site to a position lying 30 bp upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated an absence of both TATA-box and GC-box sequences; this distinguishes it from the promoters for class I, II, and III ADH genes. Sequence comparison of the mouse and human class IV ADH promoters indicated that they share a conserved region located 125-145 bp upstream of the coding region containing adjacent sequences matching the consensus binding sites for transcription factors AP-1 and C/EBP.

Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic acid synthesis.

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus--all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.

Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV alcohol dehydrogenase gene expression.

Endogenous retinoic acid (RA) has been observed in vertebrate embryos as early as gastrulation, but the mechanism controlling spatiotemporal synthesis of this important regulatory molecule remains unknown. Some members of the alcohol dehydrogenase (ADH) family catalyze retinol oxidation, the rate-limiting step in RA synthesis. Here we have examined mouse embryos for the presence of endogenous RA and expression of ADH genes. RA was not detected in egg cylinder stage embryos but was detected in late primitive streak stage embryos. Detection of class IV ADH mRNA, but not class I or class III, coincided with the onset of RA synthesis, being absent in egg cylinder embryos but present in the posterior mesoderm of late primitive streak embryos. During neurulation, RA and class IV ADH mRNA were colocalized in the craniofacial region, trunk, and forelimb bud. Class IV ADH mRNA was detected in cranial neural crest cells and craniofacial mesenchyme as well as trunk and forelimb bud mesenchyme. The spatiotemporal expression pattern and enzymatic properties of class IV ADH are thus consistent with a crucial function in RA synthesis during embryogenesis. In addition, the finding of endogenous RA and class IV ADH mRNA in the craniofacial region has implications for the mechanism of fetal alcohol syndrome.

Cloning of the mouse class IV alcohol dehydrogenase (retinol dehydrogenase) cDNA and tissue-specific expression patterns of the murine ADH gene family.

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.

Genomic structure and expression of the ADH7 gene encoding human class IV alcohol dehydrogenase, the form most efficient for retinol metabolism in vitro.

Human alcohol dehydrogenase (ADH) consists of a family of five evolutionarily related classes of enzymes that collectively function in the metabolism of a wide variety of alcohols including ethanol and retinol. Class IV ADH has been found to be the most active as a retinol dehydrogenase, thus it may participate in retinoic acid synthesis. The gene encoding class IV ADH (ADH7) has now been cloned and subjected to molecular examination. Southern blot analysis indicated that class IV ADH is encoded by a single unique gene and has no related pseudogenes. The class IV ADH gene is divided into nine exons, consistent with the highly conserved intron/exon structure of other mammalian ADH genes. The predicted amino acid sequence of the exon coding regions indicates that a protein of 373 amino acids, excluding the amino-terminal methionine, would be translated, sharing greater sequence identity with class I ADH (69%) than with classes II, III or V (59-61%). Expression of class IV ADH mRNA was detected in human stomach but not liver. This correlates with previous protein studies, which have indicated that class IV ADH is the major stomach ADH but unlike other ADHs is absent from liver. Primer extension studies using human stomach RNA were performed to identify the transcription initiation site lying 100 base pairs upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated the absence of a TATA box sequence often located about 25 base pairs upstream of the start site as well as the absence of GC boxes, which are quite often seen in promoters lacking a TATA box. The class IV ADH promoter thus differs from the other ADH promoters, which contain either a TATA box (classes I and II) or GC-boxes (class III), suggesting a fundamentally different form of transcriptional regulation.

The complete structure of human class IV alcohol dehydrogenase (retinol dehydrogenase) determined from the ADH7 gene.

A novel human alcohol dehydrogenase (ADH) gene called ADH7 has been characterized and determined to encode class IV ADH, an ADH isozyme which is very active as a retinol dehydrogenase. A nearly full-length cDNA for ADH7 was isolated from a human stomach cDNA library, and a 5' genomic clone containing exons 1 and 2 was isolated from a human genomic library. DNA sequence analysis of the cDNA and genomic clones revealed the complete coding region of the gene and the deduced full-length amino acid sequence of human class IV ADH composed of 373 amino acids following the initiator methionine. The class IV identity of the sequence was confirmed by agreement with previously determined sequences for several human stomach class IV ADH peptides. Alignment of the full-length predicted amino acid sequence of human class IV ADH with the full-length sequences of the other four known human ADH classes revealed sequence identities of 69% (class I), 59% (class II), 61% (class III), and 60% (class V). The higher sequence identity shared with human class I ADH suggests that the genes for ADH classes I and IV may have diverged from a common ancestor after the separation of the other classes, and may still share common physiological functions. Discussed is the possibility that one of these functions is retinol oxidation for the synthesis of retinoic acid, a hormone important for cellular differentiation.

Differential activity of the promoter for the human alcohol dehydrogenase (retinol dehydrogenase) gene ADH3 in neural tube of transgenic mouse embryos.

Mammalian alcohol dehydrogenase (ADH) has previously been shown to function in vitro as a retinol dehydrogenase as well as an ethanol dehydrogenase. Thus ADH participates in the conversion of retinol (vitamin A alcohol) to retinoic acid, a regulatory ligand for the retinoic acid receptor class of transcription factors. Human ADH exists as a family of isozymes encoded by seven genes, which are differentially expressed in adult liver and extrahepatic tissues, being found preferentially in the epithelial cells which are retinoid target tissues. However, human ADH expression patterns have not been analyzed in early embryonic tissues, which are known to synthesize and respond to retinoic acid such as the neural tube and limb buds. To estimate the embryonic expression pattern for one member of the human ADH family, we have constructed transgenic mouse lines carrying the human ADH3 promoter fused to the lacZ gene. ADH3-lacZ transgene expression was first noted at embryonic day 9.5 and was active in the neural tube extending from the midbrain to the spinal cord, as well as the heart and proximal regions of the forelimb buds. In day 12.5 and 13.5 embryos, ADH3 transgene expression remained in the neural tube and heart and was also observed in more distal regions of the forelimb and hindlimb buds as well as the kidney. Expression in the neural tube was highest in the ventral midline including the floor plate and showed a ventral to dorsal gradient of decreasing expression. These findings indicate that at least one human ADH isozyme may exist in the correct tissues to act as an embryonic retinol dehydrogenase catalyzing the synthesis of retinoic acid.