Cloning and characterization of the human protein kinase C-eta promoter.
Protein kinase C-eta (PKC-eta) is predominantly expressed in epithelial tissue, including lung, intestine, and skin. In skin, PKC-eta expression is limited to keratinocytes in the upper layers of the epidermis. To investigate regulation of cell type-specific expression of PKC-eta, we cloned the 5'-segment of the PKC-eta gene from a P1 genomic library. A 9.4-kilobase pair fragment encompassing the 5'-flanking region, first exon, and first intron, was localized on human chromosome 14 (14q22-23). Two major transcription initiation sites identified by reverse transcriptase polymerase chain reaction, primer extension, and S1 nuclease mapping, were located approximately 650 base pairs upstream from the translation start site. The human PKC-eta proximal promoter region lacks canonical TATA and CAAT boxes and GC-rich regions. A 1.6-kilobase pair 5'-flanking region displayed maximal promoter activity. This promoter was active in human keratinocytes but not human skin fibroblasts, in accord with endogenous PKC-eta gene expression. Stepwise 5' deletion analysis revealed the presence of adjacent regulatory regions containing silencer and enhancer elements located 1821-1702 base pairs and 1259-1189 base pairs upstream of the transcription initiation site. Deletion of the proximal PKC-eta promoter rendered the enhancer element inactive. Both the silencer and enhancer elements regulated heterologous promoters in keratinocytes but not fibroblasts. Electrophoretic mobility shift analysis demonstrated specific protein binding to Ets/heat shock factor and Ets/activator protein-1 consensus sequences in the enhancer and silencer regions, respectively. Mutations of the Ets/heat shock factor binding sites caused loss of functional enhancer activity. These data elucidate transcriptional regulation and tissue-specific expression of the PKC-eta gene.
Quan
T
T
Department of Dermatology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Fisher
G J
GJ
D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002883:Chromosomes, Human, Pair 14; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic; D020868:Gene Silencing; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000235:genetics*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D007527:Isoenzymes; C081975:protein kinase C eta; D011493:Protein Kinase C