Thymocytes control the CD4 gene differently from mature T lymphocytes

We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner.     

Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.     

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.     

Identification of a homolog of the C alpha 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin alpha 1 gene

Although four regulatory elements are known downstream of the mouse IgH alpha gene, a single enhancer homologous to hs1,2 has been thus far described downstream of each human alpha gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream of the human alpha 1 gene. Two B cell-specific regulatory elements homologous to the murine C alpha 3'/hs3 and hs1,2,3' enhancers were found, which are duplicated downstream of alpha 2. The hs1,2 element is in inverted orientation by comparison with a recently reported alpha 1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.     

Preattentive visual search and perceptual grouping in schizophrenia

Differentiated retinal pigment epithelial (RPE) cells in vivo express basal levels of FGF-5, a secreted member of the FGF gene family. RPE cells proliferate in response to pathological events, resulting in a transient increase in FGF-5 gene expression. The goal of this study is to identify cis-acting sequences in the FGF-5 gene promoter which upregulate FGF-5 gene expression when differentiated RPE cells enter the cell cycle and proliferate. In vitro cultures of RPE cells were transfected with various FGF-5 promoter/luciferase deletion constructs, using methods specifically optimized for proliferating and differentiated RPE cells. A proximal promoter/enhancer whose activity is not cell-context dependent was identified between FGF-5 sequences -314 and +48. In addition, a silencer element (-1256/-883) was identified in the distal region which is active only in differentiated RPE cells. When tested in a heterologous system, the same element had silencer activity in differentiated cells. Two small regions in the distal FGF-5 gene promoter, -1195/-1173 and -984/-967 were able to specifically bind to nuclear proteins from differentiated RPE cells but not from proliferating RPE cells as evidenced by gel mobility shift assays. Therefore, FGF-5 gene expression in the RPE may be regulated by the formation of differentiation-specific complexes.     

Sheltering of gamma-globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the A gamma-globin gene

Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.     

Restoration of the CCAAT box or insertion of the CACCC motif activates [corrected] delta-globin gene expression

Hemoglobin A2 (HbA2), which contains delta-globin as its non-alpha-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the delta-globin gene in adult erythroid cells, we first compared promoter sequences and found that the delta-globin gene differs from the beta-globin gene in the absence of an erythroid Krüppel-like factor (EKLF) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human delta-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5' flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the delta promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type delta promoter. A set of substitutions that create an EKLF binding site centered at -85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase delta-globin gene expression, with potential future clinical benefit.