A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.
SummaryDBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maxi mal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fast ing of rats for several days influences neither the ampli tude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circa dian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glu cocorticoid hormones in establishing the DBP cycle.Two genes whose mRNAs and protein products accu mulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cho lesterol synthesis; cholesterol 7-a hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid. DBP may thus be involved in regulating cho lesterol homeostasis.
We have investigated the mechanism of functional cooperativity between specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) on the human apolipoprotein CIII (apoCIII) promoter. Cotransfections in Drosophila SL2 cells that lack endogenous Sp1 or Sp1-related activities showed that HNF-4 and Sp1 synergistically transactivate the -890/+24 apoCIII promoter up to 150-fold. Synergistic transactivation required the HNF-4 binding site of the apoCIII enhancer. Deletion of part of the Ser/Thr-rich and Gln-rich domain or the C-terminal domain of Sp1 decreased, and deletion of residues 501-610 of Sp1 increased, the functional cooperativity between Sp1 and HNF-4. Physical interactions between the two factors were demonstrated by glutathione S-transferase pull-down and co-immunoprecipitation assays. The amino terminal domain of both factors and the carboxy terminal domain of Sp1 contribute to these interactions. Antagonism between HNF-4 and Sp1 was demonstrated on homopolymeric promoters containing multiple binding sites for either factor, suggesting that the synergism between the two factors occurs only when both factors are bound simultaneously to the DNA. The observed physical interactions between Sp1 and HNF-4 in the context of the apoCIII promoter may explain in part their in vitro and in vivo synergism in the transcriptional activation of the apolipoprotein A-I/apoCIII/apolipoprotein A-IV gene cluster.
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