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.     

Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. We propose that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. Our results reveal a possible reciprocal relationship in which paused Pol II influences the activator.     

Diverse effects of Stat1 on the regulation of hsp90alpha gene under heat shock

Stat1 has been known as a regulator of gene expression and a mediator of IFNgamma signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90alpha gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90alpha gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90alpha gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90alpha. These diverse effects on the hsp90alpha gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFNgamma treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy.