Year of Publication

2000

Document Type

Dissertation

College

Arts and Sciences

Department

Biochemistry

First Advisor

Kevin D. Sarge

Second Advisor

Salvatore J. Turco

Abstract

Heat shock transcription factors (HSFs) function to regulate the expression of heatshock proteins (hsps) or molecular chaperones in the cell. Mammalian cells have twowell-characterized HSFs, HSF1 and HSF2. HSF1 functions to regulate the stress-inducedexpression of hsps. The function of HSF2 appears to be in regulating hsp expressionduring development and differentiation.In this work, I describe two distinct HSF1 mRNA isoforms (HSF1-a and HSF1-b)that are generated by alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNAisoforms result from the inclusion (HSF1-a), or omission (HSF1-b), of a 66 nucleotideexon of the HSF1 gene, which encodes a 22 amino acid sequence. These results showthat the levels of the HSF1-a and HSF1-b mRNA isoforms are regulated in a tissuedependentmanner, with testis expressing predominantly the HSF1-b isoform while heartand brain express primarily the HSF1-a isoform.In addition, I describe two distinct HSF2 mRNA isoforms (HSF2-a and HSF2-b)that are generated by alternative splicing of the HSF2 pre-mRNA. The two HSF2 mRNAisoforms result from the inclusion (HSF2-a), or omission (HSF2-b), of a 54 nucleotideexon of the HSF2 gene, which encodes a 18 amino acid sequence. These results showthat the levels of the HSF2-a and HSF2-b mRNA isoforms are regulated in a tissuedependentmanner, with testis and brain expressing predominantly the HSF2-a isoformwhile heart, liver, and kidney express primarily the HSF2-b isoform. Furthermore, HSF2isoform levels are regulated both in a developmental and cell type dependent manner inthe testis. In a reporter assay, HSF2-a is a 2.6-fold better transcriptional activator thanthe HSF2-b isoform.We have demonstrated also that HSF2, but not HSF1 is a substrate for SUMO-1and SUMO-2 modification in vitro. Consistent with this, we have demonstrated thatHSF2 can interact with a portion of Ubc9, the SUMO-1 conjugating enzyme, in a twohybridassay. We have also shown that GFP-HSF2 colocalizes with SUMO-1 in discretenuclear domain structures in 7% of GFP-HSF2 expressing HeLa cells. Finally, we haveshown that lysine 82 of HSF2 is the primary site of SUMO-1 modification in vitro.

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