Ectodysplasin-A1 is sufficient to rescue both hair growth and sweat glands in Tabby mice.

Mutations in the human ectodysplasin-A (EDA) are responsible for the most common form of the ectodermal dysplasia and the defective orthologous gene in mice produces the tabby phenotype, suggesting its vital role in the development of hair, sweat glands and teeth. Among several EDA splice isoforms, the most common and the longest EDA splice isoforms, EDA-A1 and EDA-A2, differing by only two amino acids, activate NF-kappaB-promoted transcription by binding to distinct receptors, EDAR and XEDAR. The extent to which any particular isoform is sufficient for the formation of hair, sweat glands or teeth has remained unclear. Here we report that transgenic expression of the mouse EDA-A1 isoform in tabby (EDA-less) males rescued development of several skin appendages. The transgenic tabby mice showed almost complete restoration of hair growth, dermal ridges, sweat glands and molars. The number of hair follicles in the transgenic mice is the same as in wild-type; though the development of follicles and associated glands varies from indistinguishable from wild-type to smaller and/or only partially formed. These results suggest that the other EDA isoforms may not be absolutely required for skin appendage formation, but consistent with distinctive temporal and spatial expression of the EDA-A2 isoform, are likely required for appropriate timing and completeness of development. Our data provide the first direct physiological evidence that EDA-A1 is a key regulator of hair follicle and sweat gland initiation; its soluble ligand form could aid in deriving therapeutic reagents for conditions affecting hair and sweat gland formation.

Functional characterization of the promoter of the X-linked ectodermal dysplasia gene.

Anhidrotic ectodermal dysplasia (EDA) is a disorder characterized by poor development of hair, teeth, and sweat glands, and results from lesions in the X-linked EDA gene. We have cloned a 1.6-kilobase 5'-flanking region of the human EDA gene and used it to analyze features of transcriptional regulation. Primer extension analysis located a single transcription initiation site 264 base pairs (bp) upstream of the translation start site. When the intact cloned fragment or truncated derivatives were placed upstream of a reporter luciferase gene and transfected into a series of cultured cells, expression comparable with that conferred by an SV40 promoter-enhancer was observed. The region lacks a TATA box sequence, and basal transcription from the unique start site is dependent on two binding sites for the Sp1 transcription factor. One site lies 38 bp 5' to the transcription start site, in a 71-bp sequence that is sufficient to support up to 35% of maximal transcription. The functional importance of the Sp1 sites was demonstrated when cotransfection of an Sp1 expression vector transactivated the EDA promoter in the SL2 Drosophila cell line that otherwise lacks endogenous Sp1. Also, both Sp1 binding sites were active in footprinting and gel shift assays in the presence of either crude HeLa cell nuclear extract or purified Sp1 and lost activity when the binding sites were mutated. A second region involved in positive control was localized to a 40-bp sequence between -673 and -633 bp. This region activated an SV40 minimal promoter 4- to 5-fold in an orientation-independent manner and is thus inferred to contain an enhancer region.

The FixK2 protein is involved in regulation of symbiotic hydrogenase expression in Bradyrhizobium japonicum.

The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2 in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated. Bacteroids from FixJ and FixK2 mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1 mutant exhibit wild-type levels of hydrogenase activity. In beta-galactosidase transcriptional assays with NifA and FixK2 expression plasmids, the FixK2 protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2 is involved in the transcriptional activation of symbiotic hydrogenase expression. By using beta-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site.

Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum.

In-frame deletion mutagenesis was used to study the roles of two Bradyrhizobium japonicum proteins, HoxX and HoxA, in hydrogenase biosynthesis; based on their sequences, these proteins were previously proposed to be sensor and regulator proteins, respectively, of a two-component regulatory system necessary for hydrogenase transcription. Deletion of the hoxX gene resulted in a strain that expressed only 30 to 40% of wild-type hydrogenase activity. The inactive unprocessed form of the hydrogenase large subunit accumulated in this strain, indicating a role for HoxX in posttranslational processing of the hydrogenase enzyme but not in transcriptional regulation. Strains containing a deletion of the hoxA gene or a double mutation (hoxX and hoxA) did not exhibit any hydrogenase activity under free-living conditions, and extracts from these strains were inactive in gel retardation assays with a 158-bp fragment of the DNA region upstream of the hupSL operon. However, bacteroids from root nodules formed by all three mutant types (hoxX, hoxA, and hoxX hoxA) exhibited hydrogenase activity comparable to that of wild-type bacteroids. Bacteroid extracts from all of these strains, including the wild type, failed to cause a shift of the hydrogenase upstream region used in our assay. It was shown that HoxA is a DNA-binding transcriptional activator of hydrogenase structural gene expression under free-living conditions but not under symbiotic conditions. Although symbiotic hydrogenase expression is still sigma54 dependent, a transcriptional activator other than HoxA functions presumably upstream of the HoxA binding site.