Regulation of Heat Shock Transcription Factor in Drosophila

Author: Zandi, Ebrahim

Year: 1996

Degree: Dissertation (Ph.D.)

Advisor: Parker, Carl Stevens

Committee Members: Dervan, Peter B.; Parker, Carl Stevens; Campbell, Judith L.; Meyerowitz, Elliot M.

Option: Chemistry

DOI: 10.7907/hh4y-hq48

Abstract

In response to heat shock and other cellular stressors, heat shock transcription factor (HSF) stimulates transcription of heat shock genes, a set of evolutionarily highly conserved gene family whose protein products are essential for many cellular processes including cell growth and development of thermotolerance. In higher eukaryotes, at onset of heat shock, HSF protein converts from a latent state, to a DNA binding one.

Both cytological and biochemical cell fractionation show that Drosophila HSF (dHSF) is cytosolic under normal condition and nuclear after heat shock. A heat-responsive nuclear localization sequence (NLS) in the dHSF was identified, with two interrelated functions suggested. The first is found in the basic residues which are essential for nuclear entry. Mutations in these residues completely block nuclear entry. The second function identified restrains the HSF in the cytosol. Two residues were found in the NLS which when altered allowed constitutive nuclear entry of dHSF independent of heat shock. These residues may interact with a putative cellular component or with other domains of the HSF to prevent nuclear entry.

The dHSF state of oligomerization, which is regulated by heat shock, was found to be dependent upon its subcellular localization. HSF when present in the cytosolic fraction of non shocked cells is monomeric and does not bind DNA. The nuclear HSF is trimeric and binds DNA even in absence of heat shock. The same mutations in the NLS that affected the subcellular localization also affected the regulation of the oligomerization and DNA binding. The over expression of the dHSF resulted in its constitutive nuclear entry and DNA binding in the absence of heat shock suggesting the existence of a titratable inhibitory mechanism for HSF in the cytosol.

An activity which inhibited the DNA binding of the nuclear HSF in vitro was identified in the cytosolic fraction of cultured Drosophila cells and embryos. The inhibition is specific for the dHSF and is not due to a degradation of protein. This activity did not quantitatively transform trimeric HSF to the monomeric form in vitro. This and further in vitro analysis of the monomer-to-trimer transition of the dHSF suggest the existence of a multi-component complex for regulation of the dHSF's activity.

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