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Regulatory mechanisms of the heat shock response

Citation

Chen, Tianxin (Cynthia) (2002) Regulatory mechanisms of the heat shock response. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:01252012-152553827

Abstract

Although Drosophila heat shock transcription factor (dHSF) is abundant in the early embryos, it does not enter the nucleus in response to heat shock. Using the nuclear localization signal (NLS) of dHSF as bait in a yeast two-hybrid system, we identified and cloned a nuclear transport protein, Drosophila Karyopherin α3 (dKap α3). dKap α3 binds specifically to the dHSF's NLS, but not to mutant NLSs that abolish transport in vivo. The early embryo is deficient in dKap α3 protein through cycle 12, resulting in dHSF nuclear exclusion. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus, allowing the embryo to respond to heat shock. The functional domain organization of dKap α3 was mapped in detail using yeast two-hybrid analysis and immuno-fluorescence staining.

The function and specificity of the multiple Karyopherin αs found in higher eukaryotes is not understood. RNA interference was used to knock out each Kap α protein individually in Drosophila S2 cells. We found that RNA polymerase II, TATA binding protein and heat shock transcription factor were transported into the nucleus by different karyopherins, indicating that unique and non-overlapping pathways exist.

In Saccharomyces cerevisiae, the transcriptional activity of HSF is repressed at non-stress temperatures. Certain mutations of Arginine 274 in the DNA-binding domain (DBD) increase both basal and induced activities of HSF. We demonstrate that the mutations reduce the association between the DBD/oligomerization domain and the transcription activation domains. Our studies suggest that the DBD of HSF can interact with activation domains directly, and this interaction is important for the repression of HSF activity.

RNA polymerase II is bound to Drosophila Hsp70 promoters in the absence of heat shock, and paused after initiating a short transcript. Heat shock induces RNA polymerase II hyperphosphorylation and stimulates RNA polymerase II into productive elongation. We demonstrate that knocking out the expression of positive transcription elongation factor b (P-TEFb) significantly reduces Hsp70 mRN A transcription. P-TEFb is bound to HSF upon heat shock, and the complex can phosphorylate polymerase II C-terminal domain (CTD) in vitro. CTD kinase activity is inhibited by either RNAi targeting CDK9, or CDK9 inhibitor DRB.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biochemistry and Molecular Biophysics
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Parker, Carl Stevens
Thesis Committee:
  • Unknown, Unknown
Defense Date:1 April 2002
Record Number:CaltechTHESIS:01252012-152553827
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:01252012-152553827
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6780
Collection:CaltechTHESIS
Deposited By: John Wade
Deposited On:26 Jan 2012 16:35
Last Modified:26 Dec 2012 04:39

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