No abstract

We have studied the initial development of pluiipotent gut endoderm to hepatocytes using a tissue explant system from mouse embryos. We not only find cellular interactions that specify hepatic differentiation but also those that block hepatogenesis in regions of the endoderm that normally give rise to other tissues. The results implicate both positive and negative signaling in early hepatic specification. In vivo footprinting of the albumin enhancer in precursor gut endoderm shows that the transcriptionally silent but potentially active chromatin is characterized by occupancy of an HNF-3 site. Upon hepatic specification, a host of other factors bind nearby sites as the gene becomes active. Genes in pluripotent cells therefore may be marked for potential expression by entry points in chromatin, where additional factors bind during cell type specification. The findings also provide insight into the evolutionary origin of different endodermal cell types.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors central to the regulation of lipid metabolism. The SREBPs are synthesized as precursor proteins that require proteolytic processing to become transcriptionally active. Whereas the regulation of SREBP-1a and -2 cleavage by cellular sterol content is well defined, much less is known about the regulation of SREBP-1c, the predominant SREBP isoform in the liver. Both insulin and liver X receptor ␣ (LXR␣) induce SREBP-1c transcription; however, the respective roles of these factors and the mechanism responsible for proteolytic cleavage of this SREBP isoform are not known. In this study, we compare the effects of insulin and LXR agonist TO-901317 on SREBP-1c expression and transcriptional activity in isolated rat hepatocytes. We report that full induction of the mature and transcriptionally active form of SREBP-1c protein requires insulin. Although activation of LXR leads to the induction of SREBP-1c gene expression and precursor protein, it has a very poor effect in inducing the mature nuclear form of the transcription factor. This may be due to the induction of insulin-induced gene-2a mRNA and protein by LXR activation. The LXR-induced SREBP-1c precursor, however, is rapidly cleaved on acute exposure to insulin via a phosphatidylinositol 3-kinase-dependent mechanism. Finally, we show through experiments in suckling mice that this acute action of insulin to stimulate the proteolytic processing of SREBP-1c is functional in vivo.glucose homeostasis ͉ cholesterol ͉ lipogenesis ͉ hepatocytes T he sterol regulatory element-binding proteins (SREBPs) are transcription factors integral to the maintenance of lipid homeostasis. The three SREBP isoforms (SREBP-1a, -1c, and -2) have overlapping target genes and show differential expression across tissues (1). SREBP-1c is the major isoform expressed in the liver and tissues involved in energy homeostasis (2). It regulates fatty acid synthesis through selective induction of hepatic glucokinase (GK) and an array of lipogenic genes (3-6). SREBP-2 is widely expressed and primarily regulates genes involved in cholesterol biosynthesis (7). The SREBP-1a isoform, which can transactivate both lipogenic and cholesterogenic genes, is highly expressed in cell lines but has very low expression in most organs in vivo (2).The SREBPs are synthesized in the endoplasmic reticulum (ER) in the form of a precursor protein. To become transcriptionally active, the SREBP precursor must undergo proteolytic cleavage in the Golgi apparatus to liberate its N-terminal domain, which constitutes the mature transcription factor (1). Two proteins are essential to this cleavage process: SREBP cleavage-activating protein (SCAP) and insulin-induced gene (Insig). SCAP is a large integral membrane protein of the ER that interacts with newly synthesized SREBP precursor and escorts it to the Golgi apparatus (8, 9). However, SCAP can also interact with Insig, another ER protein that is deeply embedded in the membranes. Insig functions to retain...