The human IL-3 locus is regulated cooperatively by two NFAT-dependent enhancers that have distinct tissue-specific activities

The human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the -4.5-kb enhancer cooperated with the -14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at -4.1 kb in T cells, mast cells, and CD34(+) myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.     

Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen.

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

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.     

Cdx binding determines the timing of enhancer activation in postnatal duodenum

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.     

A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression

The HERV-W family contains hundreds of loci diversely expressed in several physiological and pathological contexts. A unique locus termed ERVWE1 encodes an envelope glycoprotein (syncytin) involved in hominoid placental physiology. Here we show that syncytin expression is regulated by a bipartite element consisting of a cyclic AMP (cAMP)-inducible long terminal repeat (LTR) retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism. Deletion mutant analysis showed that the ERVWE1 5' LTR contains binding sites essential for basal placental activity in the region from positions +1 to +125. The region from positions +125 to +310 represents a cAMP-responsive core HERV-W promoter active in all cell types. Site-directed mutagenesis analysis highlighted the complexity of U3 regulation. ERVWE1 placenta-specific positive (e.g., T240) and negative (e.g., G71) regulatory sites were identified, as were essential sites required for basic activity (e.g., A247). The flanking sequences of the ERVWE1 provirus contain several putative regulatory elements. The upstream HERV-H and HERV-P LTRs were found to be inactive. Conversely, the 436-bp region located between the HERV-P LTR and ERVWE1 was shown to be an upstream regulatory element (URE) which is significantly active in placenta cells. This URE acts as a tissue-specific enhancer. Genetic and functional analyses of hominoid UREs revealed large differences between UREs of members of the Hominidae and the Hylobatidae. These data allowed the identification of a positive regulatory region from positions -436 to -128, a mammalian apparent LTR retrotransposon negative regulatory region from positions -128 to -67, and a trophoblast-specific enhancer (TSE) from positions -67 to -35. Putative AP-2, Sp-1, and GCMa binding sites are essential constituents of the 33-bp TSE.