Location of a glucose-dependent response region in the rat S14 promoter

Abstract

The rat S14 gene provides an excellent model to examine the DNA sequences associated with carbohydrate regulation of hepatic gene transcription. We constructed internal deletions within 5 kilobases of the 5'-up-stream region and ligated these to a luciferase reporter gene. The constructs were transfected into primary hepatocytes and pancreatic HIT cells. In hepatocytes, an increase in the medium glucose concentration led to a parallel increase in endogenous mRNA S14 content and transfected luciferase reporter activity driven by 5 kilobases of the S14 promoter. Internal deletions of several sequences from -2706 to -285 each led to a decrease in glucose-stimulated activity, suggesting that multiple elements are necessary for the transcriptional response to glucose. Deletion from -1583 to -1069 nearly abolished the glucose effect in both cell types and delineated the carbohydrate response element (CHORE). The CHORE deletion was specific for glucose, because it did not alter the response to thyroid hormone, another known regulator of this gene. Although the CHORE sequence did not confer glucose activation to either a heterologous promoter or the basal S14 promoter (bases -285 to +19), a 5-fold enhanced response was observed when two copies of the CHORE were ligated to the first 2110 basepairs of the S14 promoter. The results suggest that the CHORE contains a carbohydrate regulatory element and operates as an enhancer in concert with other sequences within the S14 gene.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells

“…We chose to systematically study the effect of high glucose on PKCb promoter activity in synchronized A10 cells. Glucose may be exerting its effect on PKCb gene expression through a response element located in the common promoter upstream of the transcription start site, as reported with L-PK and S14 gene expression in hepatocytes (20,(33)(34)(35)(36). Since PKCb is a low copy gene, nuclear run-on assays were not a practical approach to examine transcriptional regulation.…”

Functional and gene network analyses of transcriptional signatures characterizing pre-weaned bovine mammary parenchyma or fat pad uncovered novel inter-tissue signaling networks during development

“…Although not present in the network shown in Figure 4, DGAT2 is considered to be a PPARG -target gene in non-ruminants and is related to triglyceride synthesis [44]. THRSP (potential RXRB target gene; Figure 4) is a lipogenic transcription factor and it is synergistically regulated by thyroid hormone and insulin [47,48] as well as long-term via the transcription regulator carbohydrate responsive element binding protein ( MLXIPL or ChREBP).…”

Paradoxical Triiodothyronine Suppression of S14 Transcription in Permanent Hepatic Cell Lines

“…Five kilobases or 4.3 kb of the 59-flanking region of the S14 gene were fused to pXP2 or pCAT(An), as described previously (41,42). Internal deletions were made by partial or full digestion of S14 luciferase using appropriate restriction en- …”

“…Primary hepatocytes were washed in phosphate-buffered saline and harvested as described previously (41). Permanent liver cells were harvested by the same method except 250 mL of lysis buffer was used.…”

“…zymes, described previously(41). Dr. Jack Oppenheimer(University of Minnesota, Minneapolis, MN) kindly provided the plasmid TREpal in DMMTVCAT, in which one copy of palindromic TRE was fused to DMMTVCAT (43).…”