Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.     

Dynamics of potentiation and activation: GAGA factor and its role in heat shock gene regulation.

GAGA factor (GAF) binds to specific DNA sequences and participates in a complex spectrum of chromosomal activities.Products of the Trithorax-like locus (Trl), which encodes multiple GAF isoforms, are required for homeotic gene expression and are essential for Drosophila development. While homozygous null mutations in Trl are lethal, heterozygotes display enhanced position effect variegation (PEV) indicative of the broad role of GAF in chromatin architecture and its positive role in gene expression.The distribution of GAF on chromosomes is complex, as it is associated with hundreds of chromosomal loci in euchromatin of salivary gland polytene chromosomes, however, it also displays a strong association with pericentric heterochromatin in diploid cells, where it appears to have roles in chromosome condensation and segregation. At higher resolution GAF binding sites have been identified in the regulatory regions of many genes. In some cases, the positive role of GAF in gene expression has been examined in detail using a variety of genetic, biochemical, and cytological approaches. Here we review what is currently known of GAF and, in the context of the heat shock genes of Drosophila, we examine the effects of GAF on multiple steps in gene expression.