BOOK-Post-Transcriptional Regulation of Mammalian Heat Shock

University of Kentucky UKnowledge

University of Kentucky Doctoral Dissertations

Graduate School 2000

POST-TRANSCRIPTIONAL REGULATION OF MAMMALIAN HEAT SHOCK FACTORS Michael L. Goodson University of Kentucky

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Goodson, Michael L., "POST-TRANSCRIPTIONAL REGULATION OF MAMMALIAN HEAT SHOCK FACTORS" (2000).University of Kentucky Doctoral Dissertations.Paper 256.

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ABSTRACT OF DISSERTATION Michael L. Goodson

The Graduate School

University of Kentucky

2000

POST-TRANSCRIPTIONAL REGULATION

OF MAMMALIAN HEAT SHOCK FACTORS

____________________________________

ABSTRACT OF DISSERTATION

____________________________________

A dissertation submitted in partial fulfillment of the

Requirements from the degree of Doctor of Philosophy

at the University of Kentucky

By

Michael L. Goodson

Lexington, Kentucky

Co-Director: Dr. Kevin D. Sarge, Associate Professor of Biochemistry Co-Director: Dr. Salvatore J. Turco, Professor of Biochemistry

Lexington, Kentucky

2000

ABSTRACT OF DISSERTATION

POST-TRANSCRIPTIONAL REGULATION

OF MAMMALIAN HEAT SHOCK FACTORS

Heat shock transcription factors (HSFs) function to regulate the expression of heat shock proteins (hsps) or molecular chaperones in the cell. Mammalian cells have two

well-characterized HSFs, HSF1 and HSF2. HSF1 functions to regulate the stress-induced expression of hsps. The function of HSF2 appears to be in regulating hsp expression during development and differentiation.

In this work, I describe two distinct HSF1 mRNA isoforms (HSF1-α and HSF1-β) that are generated by alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms result from the inclusion (HSF1-α), or omission (HSF1-β), of a 66 nucleotide exon of the HSF1 gene, which encodes a 22 amino acid sequence. These results show that the levels of the HSF1-α and HSF1-β mRNA isoforms are regulated in a tissue-dependent manner, with testis expressing predominantly the HSF1-β isoform while heart and brain express primarily the HSF1-α isoform.

In addition, I describe two distinct HSF2 mRNA isoforms (HSF2-α and HSF2-β) that are generated by alternative splicing of the HSF2 pre-mRNA. The two HSF2 mRNA isoforms result from the inclusion (HSF2-α), or omission (HSF2-β), of a 54 nucleotide

exon of the HSF2 gene, which encodes a 18 amino acid sequence. These results show

that the levels of the HSF2-α and HSF2-β mRNA isoforms are regulated in a tissue-dependent manner, with testis and brain expressing predominantly the HSF2-α isoform

while heart, liver, and kidney express primarily the HSF2-β isoform. Furthermore, HSF2 isoform levels are regulated both in a developmental and cell type dependent manner in

the testis. In a reporter assay, HSF2-α is a 2.6-fold better transcriptional activator than

the HSF2-β isoform.

We have demonstrated also that HSF2, but not HSF1 is a substrate for SUMO-1

and SUMO-2 modification in vitro. Consistent with this, we have demonstrated that

HSF2 can interact with a portion of Ubc9, the SUMO-1 conjugating enzyme, in a two-

hybrid assay. We have also shown that GFP-HSF2 colocalizes with SUMO-1 in discrete nuclear domain structures in 7% of GFP-HSF2 expressing HeLa cells. Finally, we have shown that lysine 82 of HSF2 is the primary site of SUMO-1 modification in vitro.

____________________________________

____________________________________

POST-TRANSCRIPTIONAL REGULATION

OF MAMMALIAN HEAT SHOCK FACTORS

By

Michael L. Goodson

____________________________________

Co-Director of Research

____________________________________

Co-Director of Research

____________________________________

Director of Gratudate Studies

____________________________________

RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor's degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be

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the signature of each user.

Name Date

DISSERTATION

Michael L. Goodson

The Graduate School University of Kentucky

2000

POST-TRANSCRIPTIONAL REGULATION

OF MAMMALIAN HEAT SHOCK FACTORS

____________________________________

DISSERTATION

____________________________________

A dissertation submitted in partial fulfillment of the

Requirements from the degree of Doctor of Philosophy

at the University of Kentucky

By

Michael L. Goodson

Lexington, Kentucky

Co-Director: Dr. Kevin D. Sarge, Associate Professor of Biochemistry Co-Director: Dr. Salvatore J. Turco, Professor of Biochemistry

Lexington, Kentucky

2000

Acknowledgments

First, I would like to acknowledge the contribution of Dr. Kevin Sarge, my mentor, by whose efforts, I have learned a great deal about the scientific process. Needless to say (though I still am saying it), this work could not have happened without his support. Secondly, I would like to recognize the support of my dissertation committee: Dr. Sam Turco, Dr. Mark Kindy, Dr. Wendy Katz, and Dr. Brett Spear. Their advice has been instrumental in my work. I would also like to thank my outside examiner Dr. Tae Ji, for his comments on my dissertation during the thesis defense process.

I would also very much like to acknowledge our department’s graduate secretary, Carol Fowler. Every biochemistry graduate student reaps the benefits of Carol’s tireless efforts to make to graduate process as pleasant as possible. When she retires next month she will be sorely missed.

Also, I would like to acknowledge our collaborators. Dr. Michael Matunis performed all of the SUMO-1 in vitro modification assays. In addition, he provided many plasmids, the SUMO-1 antibody, and a great deal of SUMO-1 expertise. Without his help, Chapter 3 of this dissertation would not have been possible. Dr. Joana Desterro and Dr. Ronald Hay provided several SUMO-1 plasmids, including the GST-SUMO-1 expression plasmid. Dr. Yongho In and Dr. Okkyong Park-Sarge provided the Ubc9 yeast two hybrid plasmid and initially discovered the Ubc9/HSF2 interaction. Finally, Dr. Sindey Whiteheart provided expertise with immunofluorescent microscopy, as well as the microscope. Thanks, Wally.

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Finally, I would like to thank my family. My parents have always been supportive of my education. I’m still not leaving college, Mom. Most of all, however, I would like to thank God from whom all of life’s blessings flow, and to thank my wife Brenda, the greatest of all blessings. She has been both a great scientist and a great friend. To this work she has contributed innumerable ideas as well as editorial comments. She has also always been my solace throughout this process. Brenda, you are my colleague, my friend, my bulwark, my lover, my soul mate. You give my life meaning. I love you, and I dedicate this work to you.

And, because every dissertation should have a little Latin in it. . .

"Probae esti in segetem sunt deteriorem datae fruges, tamen ipsae suaptae enitent."

-- Accius, Atreus

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Table of Contents Acknowledgments (iii)

List of Tables (vii)

List of Figures (viii)

Chapter 1 Background and Introduction (1)

Transcription factors, regulators of eukaryotic RNA synthesis (1)

RNA splicing—removing garbage or creating persity? (4)

The cellular stress response (12)

Spermatogenesis, the process of germ cell maturation (18)

Chapter 2 Alternative Splicing Isoforms of HSF1 and HSF2 (29)

Introduction (29)

Materials and Methods (39)

Experimental Animals (39)

RT-PCR Analysis (39)

Isolation and Cloning of HSF1 cDNA and Genomic DNA Sequences (41)

Western Blot (Immunoblot) and Gel Mobility Shift Analysis (42)

HSF2 Transfection of NIH 3T3 Cells and Luciferase Assays (42)

Results (43)

Tissue distribution of HSF1 mRNA isoforms (43)

Tissue distribution of HSF2 mRNA isoforms (44)

Cloning of HSF1 cDNA isoforms (45)

Cloning of HSF2 cDNA isoforms (46)

Cloning of the HSF1 genomic DNA from the splice variant region (46)

HSF1-α splicing creates a fifth potential leucine zipper (47)

Cloning of the HSF2 genomic DNA from the splice variant region (48)

Developmental Regulation of HSF2 mRNA Splicing (49)

Increased transcriptional activity of the HSF2-α isoform (49)

Discussion (75)

HSF1 alternative splicing—implications for differential stress response activation.75 HSF2 alternative splicing—implications for spermatogenic gene regulation (76)

Chapter 3 SUMO-1 Modification of HSF2 (79)

Introduction (79)

Materials and Methods (93)

Plasmid DNA Construction (93)

SUMO-1 Consensus Site Pattern Matching (94)

Site Directed Mutagenesis of HSF2 (95)

Yeast Transformation and the Two-Hybrid Assay (95)

In vitro SUMO-1 Modification Assay (97)

Recombinant Protein Expression (97)

Recombinant Protein Purification (98)

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Transient Transfection of HeLa Cells (100)

Immunofluorescent Microscopy (101)

Results (102)

Two Hybrid Analysis of the HSF2/Ubc9 interaction (102)

In vitro SUMO-1 modification of HSF2 (106)

Nuclear colocalization of SUMO-1 and GFP-HSF2 (108)

Identification of the SUMO-1 modification site in HSF2 (110)

Discussion (130)

Chapter 4 Discussion and Future Directions (132)

The functional difference between HSF1-α and HSF1-β (132)

The possibility of stress induced SUMO modification of HSF1 (133)

The role of HSF2-α and HSF2-β in spermatogenesis (134)

Other functions of HSF2 (134)

The regulation of the SUMO-1 modification of HSF2 (135)

The 26S proteosome and SUMO modification of HSF2 (136)

Appendix (138)

Appendix A: List of Abbreviations (138)

References (139)

Vitae (152)

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List of Tables

Table 1. Quantification of GFP-HSF2 nuclear domain staining (110)

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List of Figures

Figure 1.1: Schematic representation of the RNA splicing reaction (7)

Figure 1.2: Schematic representation of alternative splicing (11)

Figure 1.3: Schematic diagram of the cellular stress response (15)

Figure 1.4: Schematic diagram of spermatogenesis (22)

Figure 1.5: Diagram of one cycle of spermatogenic stages (27)

Figure 2.1: Human and mouse HSF DNA and protein sequence alignments (31)

Figure 2.2: RT-PCR analysis of HSF1 mRNA isoforms in mouse tissues (52)

Figure 2.3: Western blot analysis of HSF2 protein from mouse tissues (54)

Figure 2.4: RT-PCR analysis of HSF2 mRNA isoforms in mouse tissues (56)

Figure 2.5: Nucleotide and deduced amino acid sequences of HSF1 mRNA isoform cDNAs (58)

Figure 2.6: Nucleotide and deduced amino acid sequences of HSF2 mRNA isoform cDNAs (60)

Figure 2.7: Sequence of HSF1 gene regions corresponding to alternative splice junctions.

(62)

Figure 2.8: Schematic representation of HSF1 mRNA alternative splicing (64)

Figure 2.9: Novel leucine zipper motif in HSF1-α (66)

Figure 2.10: Sequence of HSF2 gene regions corresponding to alternative splice junctions (68)

Figure 2.11: Schematic representation of HSF2 mRNA alternative splicing (70)

Figure 2.12: RT-PCR analysis of HSF2 isoforms during testis development (72)

Figure 2.13: Reporter gene analysis of HSF2-α and HSF2-β isoforms (74)

Figure 3.1: Schematic representation of the ubiquitination cycle (81)

Figure 3.2: Schematic representation of the SUMO-1 modification cycle (86)

Figure 3.3: Schematic diagram of the yeast two hybrid assay (105)

Figure 3.4: Two-hybrid analysis of the HSF2/Ubc9 interaction (113)

Figure 3.5: In vitro SUMO-1 and SUMO-2 modification of HSF2 (115)

Figure 3.6: In vitro SUMO-1 modification analysis of HSF1 (117)

Figure 3.7: Colocalization of GSP-HSF2 and SUMO-1 (119)

Figure 3.8: Unique localization of GFP-HSF2 (121)

Figure 3.9: Purification of recombinant HSF1 and SUMO-1 (123)

Figure 3.10: Preadsorbed control for SUMO-1 Immunofluorescent Staining (125)

Figure 3.11: Consensus SUMO-1 modification site analysis of HSF2 (127)

Figure 3.12: In vitro modification analysis of HSF2 mutants (129)

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Chapter 1

Background and Introduction

T RANSCRIPTION FACTORS, REGULATORS OF EUKARYOTIC RNA SYNTHESIS

Each cell in a multicellular organism has DNA with exactly the same sequence as every other cell in that organism, yet the cells of that organism are highly perse both in function and morphology. With only a few small exceptions, such as gene rearrangement

in immune cells, germ cells, transposons, and random mutations, this is true for every metazoan. How then does an organism generate this cellular persity from identical

genetic material? The answer to this lies in the pattern of gene expression. Different

cells express different genes at different levels. Therefore, an organism must carefully regulate the expression of its genes. One major mechanism for controlling gene

expression is by regulating transcription of DNA into RNA (Maniatis et al., 1987).

Eukaryotic genes are transcribed by one of three RNA polymerases. RNA polymerase I transcribes ribosomal RNA. RNA polymerase III transcribes small RNA molecules such as the 5S ribosomal RNA and transfer RNA. RNA polymerase II transcribes RNA from genes that will be translated into protein, called messenger RNA (Chambon, 1975; Geiduschek and Tocchini-Valentini, 1988; Sentenac, 1985; Sollner-

Webb and Tower, 1986). In eukaryotes, RNA polymerases are large multi-subunit

protein complexes with masses of 500 kDa or more. Unlike in prokaryotes and viruses,

the eukaryotic RNA polymerases do not directly recognize DNA sequences. Rather

1

2 DNA binding proteins, called transcription factors, bind to specific sequences in the promoter regions of genes and thereby recruit the RNA polymerase complexes (Brown, 1984; Workman and Roeder, 1987).

Promoter regions are transcriptional regulatory sequences of genes that can be pided into two categories proximal promoter elements and distal enhancer elements.

The basal promoter elements contain sequences such as GAGA elements, the TATA box,

or the initiator (Inr) motif. Basal promoter elements are highly context sensitive and

must be located near the transcription start site (Atchison, 1988; Maniatis et al., 1987; McKnight and Kingsbury, 1982). For example, in genes that contain one, the TATA box

is always located approximately 30 bp upstream of the start site. In contrast, enhancer elements are often found several kb upstream of the transcription start site. They can also

be found several kilobases upstream of the gene, downstream of the gene, or within the transcribed region of the gene. Enhancer regions usually contain binding sites for

multiple regulatory proteins and are normally modular. This modular quality means that enhancers can often be moved to different locations within the promoter region of a gene,

or within the context of a completely different basal promoter and gene (as in the case of

a reporter gene assay) (Atchison, 1988; Emerson et al., 1987; Evans et al., 1988; Jones et al., 1988; Nomiyama et al., 1987).

Similarly transcription factors can be pided into two categories: i) general transcription factors, which bind to basal promoter elements in nearly all genes and to the RNA polymerase complex, and ii) transcription enhancers and repressors which bind to enhancer elements. For the purpose of this introduction, I will specifically refer to

general transcription factors and will often refer to transcription enhancers and repressors

3 as transcription factors. All three RNA polymerases have general transcription factors (TFI, TFII, and TFIII) for binding to promoters and regulating transcription of their respective genes. In this introduction, I will limit discussion to RNA polymerase II transcription factors.

General transcription factors bind to the basal promoter region of most genes forming a stable complex on the DNA and recruiting the RNA polymerase. Examples of general transcription factors include TFIIA, TFIIB, TFIID (which includes the TATA binding protein, TBP), TFIIE, and TFIIH and GAGA factors (Burley and Roeder, 1996; Orphanides et al., 1996; Roeder, 1996). These factors are expressed in all tissues, and therefore cannot account for the perse patterns of gene expression found in the body.

Transcription enhancers and repressors, which bind to sequences in the enhancer region, are much more perse in composition, function, and expression than the general transcription factors. Heat shock factors (HSFs) are considered transcription enhancers. Transcription enhancers (or repressors) bind to DNA and modulate transcription by

several mechanisms. Some function by bending DNA and changing the proximity to

other elements (Ogbourne and Antalis, 1998). Others function by interacting with the general transcription factors or the RNA polymerase and modulating the function of these components. Still others interact with other transcription enhancers or repressors to modulate an effect synergistically (Evans, 1988; Schulman et al., 1995). Transcription enhancers and repressors are particularly interesting because they are often functionally regulated (Verrijzer and Tjian, 1996). Regulation of transcription factors may occur by regulation of transcription factor expression, by interaction with a cellular factor or

ligand, as in the steroid hormone receptors, or by modification by a receptor or receptor

4 mediated signal transduction cascade, as in STATs, fos, or jun. To this already complex paradigm of multiple transcription factors, each regulated in its own unique fashion, we

can add that most transcription factors bind as multimers (Ap-1, RXR, T3R, VDR). The composition of these multimeric transcription factor complexes often dictates DNA

binding specificity and the functional consequence of binding—whether the complex activates or represses transcription (Evans, 1988; Umesono and Evans, 1989; Umesono et al., 1991). Also, many transcription factors interact in a regulated fashion with other

cellular factors that can modulate transcriptional activity. Such layers of regulation can

create the tremendous persity of gene expression necessary for a multicellular organism (Chen, 1999).

RNA SPLICING—REMOVING GARBAGE OR CREATING DIVERSITY?

As described previously, eukaryotic genes are transcribed by one of three RNA polymerases. Of these, only RNA polymerase II transcribes genes that will be translated

into proteins. The mRNA transcribed from RNA polymerase II is modified at the 5’ end

by the addition of a unique cap structure—7-methyl-guanosine in a 5’ to 5’ triphosphate linkage—called the 5’ cap (Shatkin, 1987). The 3’ end of the RNA is also modified by

the addition of a series of non-encoded adenosine residues called the poly-A tail. Only messenger RNA contains a 5’ cap and a ploy-A tail (Sisodia et al., 1987; Smale and

Tjian, 1985).

In addition to 5’capping and poly-A tailing, eukaryotic mRNA, particularly

mRNA from metazoans, requires further processing. The genes encoding proteins in

5 higher eukaryotes contain both coding sequences referred to as exons and intervening sequences referred to as introns. The process of removing the introns in pre-mRNA and joining the exons to form mature mRNA is called RNA splicing (Chambon, 1981; Crick, 1979; Perry, 1981). A large macromolecular complex called the spliceosome, which contains four small nuclear ribonuceoproteins (snRNPs) U1, U2, U5, and U4/U6, usually carries out the splicing reaction (Figure 1.1) (Dreyfuss et al., 1988; Guthrie and Patterson, 1988; Osheim et al., 1985; Samarina et al., 1966; Steitz, 1988).

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