The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter.

The regulatory region of Drosophila melanogaster hsp26 includes a positioned nucleosome located between the two DNase I hypersensitive (DH) sites that encompass the critical heat shock elements (HSEs). To test the role of this nucleosome in regulated expression, transgenic flies containing hsp26-lacZ fusion genes with alterations in the nucleosome-associated region have been generated. The positioned nucleosome is associated with a DNA sequence that does not itself contain any critical regulatory elements for heat shock-inducible expression. The nucleosome-associated sequence can be deleted, reversed, duplicated or replaced by a random sequence with no significant effect on DH site formation and gene expression. Analyses of hsp26 and hsp70 transgenes with spacing changes within the promoter region indicate that the location of the (CT)n.(GA)n elements dictates the location of DH site formation. Wrapping the DNA between the regulatory elements around a nucleosome is as effective for gene expression as placing the regulatory elements close to each other. A loss of inducible gene expression was observed when the nucleosome-associated DNA was replaced with sequences which appear to misdirect nucleosome placement. The results indicate considerable flexibility in the spacing between DH regulatory sites.     

GAGA factor and the TFIID complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter

The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.     

Multiple, compensatory regulatory elements specify spermatocyte-specific expression of the Drosophila melanogaster hsp26 gene.

The hsp26 gene of Drosophila melanogaster is expressed in six tissues during development and in a tissue-general response to heat shock. To be able to compare tissue-specific and heat-induced mechanisms of hsp26 expression, we have begun an analysis of the sequences involved in the spermatocyte-specific expression of the hsp26 gene by using germ line transformation. hsp26 mRNA synthesized in the spermatocytes has the same start site as sites previously demonstrated for nurse cell-specific and heat-induced mRNAs. Three regions of the hsp26 gene (nucleotides -351 to -135, -135 to -85, and +11 to +632) were able to stimulate spermatocyte-specific expression when fused with promoter sequences (nucleotides -85 to +11) that alone were insufficient to stimulate expression. These stimulatory regions appear to contain elements that provide redundant functions. While each region was able to stimulate expression independently, the deletion of any one region from a construct was without consequence as long as another compensatory region(s) was still present. There must reside, at a minimum, two independent spermatocyte-specifying elements within the sequences that encompass the three stimulatory regions and the promoter. At least one element is contained within sequences from -351 to -48. This region, in either orientation, can stimulate spermatocyte-specific expression from a heterologous promoter. A second element must reside in sequences from -52 to +632, since these sequences are also sufficient to direct spermatocyte-specific expression.     

Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.

Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.     

Several hundred base pairs upstream of Drosophila hsp23 and 26 genes are required for their heat induction in transformed flies.

We have used the P-element-mediated transformation of Drosophila germ line to study the 5' DNA sequences involved in the thermal inducibility of the genes for heat shock proteins hsp23 and 26. The results are strikingly different from those previously obtained in heterologous systems. For hsp23, each successive shortening of the promoter region from 618 to 402, 321 and 263 bp clearly decreased the expression. A construct with only 149 bp was not inducible at all. For hsp26, all the regulatory elements appear to be clustered in the first 350 bp upstream from the cap site. Clones with 171 bp showed a 4- to 10-fold decrease in induction depending on the transformed line, and those with only 52 bp were not expressed. The results suggest that at least three Pelham consensus sequences are required for the full expression of these two genes. The direct involvement of one of these consensus sequences has been assessed: a 6-bp deletion within the proximal element of the hsp26 gene strongly reduced its inducibility. Our results also indicate that X-linked hsp genes exhibit either partial dosage compensation or none at all.     

The capacity to form H-DNA cannot substitute for GAGA factor binding to a (CT)n*(GA)n regulatory site

Previous studies of the Drosophila melanogaster hsp26 gene promoter have demonstrated the importance of a homopurine·homopyrimidine segment [primarily (CT)_n·(GA)_n] for chromatin structure formation and gene activation. (CT)_n regions are known to bind GAGA factor, a dominant enhancer of PEV thought to play a role in generating an accessible chromatin structure. The (CT)_n region can also form an H-DNA structure in vitro under acidic pH and negative supercoiling; a detailed map of that structure is reported here. To test whether the (CT)_n sequence can function through H-DNA in vivo, we have analyzed a series of hsp26-lacZ transgenes with altered sequences in this region. The results indicate that a 25 bp mirror repeat within the homopurine·homopyrimidine region, while adequate for H-DNA formation, is neither necessary nor sufficient for positive regulation of hsp26 when GAGA factor-binding sites have been eliminated. The ability to form H-DNA cannot substitute for GAGA factor binding to the (CT)_n sequence.     

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.