Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.     

Differentiation-dependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma.

Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb -2.8 to -1.0 of the marine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb -2.5 to -2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor gamma (PPARgamma) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR gamma antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR-gamma mRNA levels increase correspondingly. Although forced expression of PPAR gamma and retinoid X receptor alpha activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR gamma is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.     

Characterization of a cardiac-specific enhancer, which directs {alpha}-cardiac actin gene transcription in the mouse adult heart

Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5' to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the alpha-cardiac actin locus.     

Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.     

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.