Frederic Bone

The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation

Human involucrin (hINV) is a keratinocyte protein that is expressed in the suprabasal compartment of the epidermis and other stratifying surface epithelia. Involucrin gene expression is initiated early in the differentiation process and is maintained until terminal cell death. The distal regulatory region (DRR) is a segment of the hINV promoter (nucleotides −2473/−1953) that accurately recapitulates the normal pattern of suprabasal (spinous and granular layer) expression in transgenic mouse epithelia. To identify sequences that mediate expression at specific stages of differentiation, we divided the DRR into two segments, a 376 nucleotide upstream region (DRR−2473/−2100) and a 147 nucleotide downstream region (DRR−2100/−1953), and evaluated the ability of these sequences to drive expression in transgenic mice. The DRR−2473/−2100 segment drives expression at a level comparable to that observed for the DRR, but expression is restricted to the upper granular layers (i.e., no spinous layer expression). In contrast, the DRR−2100/−1953 segment does not drive expression. However, reassembling the DRR restores the complete range of expression. These results suggest that two distinct, spatially-separate elements are required to specify the complete differentiation-dependent program of involucrin gene expression. To identify specific transcription factor binding sites involved in this regulation, we mutated an activator protein-1 binding site, AP1-5, located within DRR−2473/-2100 segment. This site binds AP1 transcription factors present in mouse epidermal extracts, and its mutation eliminates appropriate hINV expression. This result suggests that AP1 factors participate as components of a multi-component transcription factor complex that is required for regulation.

Regulation of Glucose-6-phosphatase Gene Expression in Cultured Hepatocytes and H4IIE Cells by Short-chain Fatty Acids ROLE OF HEPATIC NUCLEAR FACTOR-4α

Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood. Here we investigated the effects of short-chain fatty acids on the expression of this gene in primary cultures of rat hepatocytes and H4IIE hepatoma cells. Propionate, butyrate, valerate, and caproate induced severalfold increases in the expression of Glc-6-Pase mRNA. In reporter gene assays, propionate, valerate, caproate, and also octanoate increased Glc-6-Pase promoter activity by 6-16-fold. Butyrate, by itself, had little or no effect on promoter activity, but it induced a robust increase (45-fold) in promoter activity in cells co-transfected with a plasmid expressing the transcription factor HNF-4α (α isoforms of hepatic nuclear factor 4). HNF-4α also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4α abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4α in the transcription process studied here. In cells transfected with HNF-4α, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4α. Region-751/-466 of this promoter contains seven putative HNF-4α-binding motifs. Binding of HNF-4α to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4α is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.