alpha(1)-fetoprotein transcription factor (FTF)/liver receptor homolog-1 (LRH-1) is an essential lipogenic regulator.

alpha(1)-Fetoprotein transcription factor (FTF), also known as liver receptor homolog 1 (LRH-1) is highly expressed in the liver and intestine, where it is implicated in the regulation of cholesterol, bile acid and steroid hormone homeostasis. FTF is an important regulator of bile acid metabolism. We show here that FTF plays a key regulatory role in lipid homeostasis including triglyceride and cholesterol homeostasis. FTF deficient mice developed lower levels of serum triglyceride and cholesterol as a result of lower expression of several hepatic FTF target genes. Chenodeoxycholic acid repressed FTF expression resulting in a decrease in serum triglyceride in wild-type mice. The absence of chenodeoxycholic acid-mediated repression in FTF(+/-) mice demonstrated the essential role of FTF in triglyceride metabolism. Taken together, our results identify the nuclear receptor FTF as a central regulator of lipid metabolism.

Regulation of sperm gene expression by the GATA factor ELT-1.

Cell fate specification is mediated primarily through the expression of cell-type-specific genes. The regulatory pathway that governs the sperm/egg decision in the hermaphrodite germ line of Caenorhabditis elegans has been well characterized, but the transcription factors that drive these developmental programs remain unknown. We report the identification of ELT-1, a GATA transcription factor that specifies hypodermal fate in the embryo, as a regulator of sperm-specific transcription in the germ line. Computational analysis identified a conserved bipartite sequence element that is found almost exclusively in the promoters of a number of sperm genes. ELT-1 was recovered in a yeast one-hybrid screen for factors that bind to that sperm consensus site. In vitro assays defined the sperm consensus sequence as an optimal binding site for ELT-1. We determined that expression of elt-1 is elevated in the sperm-producing germ line, and that ELT-1 is required for sperm function. Deletion of the ELT-1 binding site from a sperm promoter abrogates sperm-specific expression of a reporter transgene. This work demonstrates a role for the ELT-1 transcription factor in sperm, and provides a critical link between the germ line sex determination program and gamete-specific gene expression.

Critical contact residues that mediate polymerization of nematode major sperm protein.

The polymerization of protein filaments provides the motive force in a variety of cellular processes involving cell motility and intracellular transport. Regulated assembly and disassembly of the major sperm protein (MSP) underlies amoeboid movement in nematode sperm, and offers an attractive model system for characterizing the biomechanical properties of filament formation and force generation. To that end, structure-function studies of MSP from the nematode Caenorhabditis elegans have been performed. Recombinant MSP was purified from Escherichia coli using a novel affinity chromatography technique, and filament assembly was assessed by in vitro polymerization in the presence of polyethylene glycol. Prior molecular studies and structure from X-ray crystallography have implicated specific residues in protein-protein interactions necessary for filament assembly. Purified MSP containing substitutions in these residues fails to form filaments in vitro. Short peptides based on predicted sites of interaction also effectively disrupt MSP polymerization. These results confirm the structural determination of intermolecular contacts and demonstrate the importance of these residues in MSP assembly.

Regulation of group VIA phospholipase A2 expression by sterol availability.

Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) and the group VIA calcium-independent phospholipase A2 (iPLA2). CTalpha expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA2 catalytic activity, mRNA, and protein were induced by between two- and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA2, an analysis of the 5' flanking region suggested that the iPLA2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA2 activity and expression compared to wild-type cells. These data suggest that iPLA2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes.

Differential effects of sterol regulatory binding proteins 1 and 2 on sterol 12 alpha-hydroxylase. SREBP-2 suppresses the sterol 12 alpha-hydroxylase promoter.

The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has implications for atherosclerosis, cholesterol gallstone formation, and some lipid storage diseases. Sterol 12 alpha-hydroxylase (12 alpha-hydroxylase) is required for cholic acid biosynthesis. The alpha(1)-fetoprotein transcription factor FTF is crucial for the expression and the bile acid-mediated down-regulation of 12 alpha-hydroxylase. Cholesterol, on the other hand, down-regulates expression of the 12 alpha-hydroxylase gene. In this study, we show that the two sterol regulatory binding proteins (SREBPs) have opposite effects on the 12 alpha-hydroxylase promoter. SREBP-1 activated the 12 alpha-hydroxylase promoter, as it does with many other cholesterol-regulated genes. In contrast, SREBP-2 suppressed 12 alpha-hydroxylase promoter activity. SREBP-1 mediates the cholesterol-down-regulation of 12 alpha-hydroxylase promoter by binding to two inverted sterol regulatory elements found approximately 300 nucleotides from the transcriptional initiation site. SREBP-2 mediated suppression of 12 alpha-hydroxylase without binding to its promoter. Data are presented suggesting that SREBP-2 suppresses the 12 alpha-hydroxylase promoter by interacting with FTF. This is the first report of a promoter responding oppositely to two members of the SREBP family of transcription factors. These studies provide a novel function and mode of action of a SREBP protein.

The role of alpha1-fetoprotein transcription factor/LRH-1 in bile acid biosynthesis: a known nuclear receptor activator that can act as a suppressor of bile acid biosynthesis.

Two key regulatory enzymes in the bile acid biosynthesis pathway are cholesterol 7alpha-hydroxylase/CYP7A1 (7alpha-hydroxylase) and sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase). It has been shown previously that hepatocyte nuclear factor-4alpha (HNF-4) and the alpha(1)-fetoprotein transcription factor (FTF) are activators of 7alpha-and 12alpha-hydroxylase transcription and that the small heterodimer partner (SHP) suppresses bile acid biosynthesis by heterodimerizing with FTF. However, the role of FTF in bile acid biosynthesis has been studied only in tissue culture systems. In heterozygous FTF knockout mice, 7alpha- and 12alpha-hydroxylase genes were expressed at 5-7-fold higher levels than in wild-type mice, an apparent direct contradiction to previous in vitro observations. This higher expression of the 7alpha- and 12alpha-hydroxylase genes resulted in a 33% higher bile acid pool in their gallbladders, bile more enriched in cholic acid, and a 13% decrease in plasma cholesterol levels. Adenovirus-mediated FTF overexpression in wild-type mice resulted in 10-fold lower expression of the 7alpha- and 12alpha-hydroxylase genes and up to 8-fold higher SHP expression, highlighting the dual role that FTF plays in different promoters. Shorter overexpression times still resulted in lower 7alpha- and 12alpha-hydroxylase expression, but unchanged SHP expression, suggesting that two different mechanisms are involved in the FTF-mediated suppression of 7alpha- and 12alpha-hydroxylase expression. This FTF-mediated suppression of the expression of two bile acid biosynthesis genes resulted in a 3-fold lower rate of bile acid synthesis in a rat bile fistula animal model. Based on these observations and on protein binding studies performed in vitro and by chromatin immunoprecipitation, we hypothesize that FTF has two synergetic effects that contribute to its role in bile acid biosynthesis: 1) it has the ability to activate the expression of SHP, which in turn heterodimerizes and suppresses FTF transactivation activity; and 2) it occupies the FTF/HNF-4 recognition site within the 7alpha- and 12alpha-hydroxylase promoters, which can otherwise be occupied by a factor (HNF-4) that cannot be suppressed by SHP.

Suppression of cholesterol 7alpha-hydroxylase transcription and bile acid synthesis by an alpha1-antitrypsin peptide via interaction with alpha1-fetoprotein transcription factor.

alpha1-Antitrypsin (alpha1-AT) is a serum protease inhibitor that is synthesized mainly in the liver, and its rate of synthesis markedly increases in response to inflammation. This increase in alpha1-AT synthesis results in an increase in peptides, like its carboxyl-terminal C-36 peptide (C-36), resulting from alpha1-AT cleavage by proteases. Atherosclerosis is a form of chronic inflammation, and one of the risk factors is elevated plasma cholesterol levels. Because of the correlation between atherosclerosis, plasma cholesterol content, inflammation, and alpha1-AT rate of synthesis, we investigated the effect of the C-36 serpin peptide on hepatic bile acid biosynthesis. We discovered that C-36 is a powerful and specific transcriptional down-regulator of bile acid synthesis in primary rat hepatocytes, through inhibition of the cholesterol 7alpha-hydroxylase/CYP7A1 (7alpha-hydroxylase) promoter. Mice injected with the C-36 peptide also showed a decrease in 7alpha-hydroxylase mRNA. A mutated but very similar peptide did not have any effect on 7alpha-hydroxylase mRNA or its promoter. The sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase) promoter is also down-regulated by the C-36 peptide in HepG2 cells but not by the mutated peptide. The DNA element involved in the C-36-mediated regulation of 7alpha- and 12alpha-hydroxylase promoters mapped to the alpha1-fetoprotein transcription factor (FTF) site in both promoters. The C-36 peptide prevented binding of FTF to its target DNA recognition site by direct interaction with FTF. We hypothesize that the C-36 peptide specifically interacts with FTF and induces a conformational change that results in loss of its DNA binding ability, which results in suppression of 7alpha- and 12alpha-hydroxylase transcription. These results suggest that peptides derived from specific serum proteins may alter hepatic gene expression in a highly specific manner.

Genomic organization and transcriptional analysis of the human l-glutaminase gene.

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18 kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter-luciferase constructs indicated that the core promoter is located between nt -141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and -87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.