Faculty Bibliography

The promoters of epidermal keratin genes, K5, K6, and K10 were cloned and their functions compared with that of the previously described promoter of the K14 keratin gene in non-epithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia. The four promoters were functional only in epithelial cells. Although the promoter for the basal cell-specific, acidic-type K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper-proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on non-epidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences

In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters

Two regions of human genomic DNA, each containing several keratin genes, were isolated and partially sequenced. The keratin genes are inactive, having suffered deleterious mutations. Both regions contain at least four keratin genes arranged in a head-to-tail orientation including a pseudogene for keratin K#16. Within each segment there are two keratin genes in close linkage with only 1.5 kb of DNA between them. Sequence comparison of the two regions showed 98.9% identity in both the coding and the intronic segments of the pseudogenes. The pseudogenes show 94% identity to their functional counterparts. Southern hybridization analysis showed that the segments are paralogous, not allelic. The regions are products of two independent, recent duplication events. The first occurred approximately 24 million years ago, after the separation of primates from the rhesus/baboon line. The second is specific for the human lineage, having occurred approximately 3.8 million years ago. Analysis of the genomic DNAs of primates showed the presence of only one of the regions in the DNAs of gibbon and gorilla, while rhesus monkey and baboon were missing both copies. We conclude that the human keratin genes are still actively evolving, with new duplications having occurred as recently as after the separation of human and gorilla ancestors