Role and regulatory mechanisms of heat shock factor 2

9 INTRODUCTION . 10 REVIEW OF THE LITERATURE . 11 1. BASIC PRINCIPLES OF TRANSCRIPTION IN EUKARYOTES . 11 1.1. Transcriptional initiation and regulatory elements . 11 1.2. Transcriptional activators and repressors . 13 1.3. RNAP II and the elongation phase of transcription. 14 1.4. Regulation of transcription at the level of chromatin . 14 1.5. Genome organization as a means to regulate transcription . 15 2. microRNAs AS REGULATORS OF GENE EXPRESSION . 17 2.1. miRNA biogenesis . 18 2.2. miRNA mechanisms of action . 19 2.3. Biological functions of miRNAs . 21 2.3.1. miRNAs in spermatogenesis . 21 2.3.2. miRNAs in disease . 22 2.4. The miR-17~92 cluster . 22 2.4.1. The miR-17~92 cluster in cancer . 23 2.4.2. The miR-17~92 cluster in development . 24 2.4.3. miR-18 . 25 3. HEAT SHOCK FACTORS AS TRANSCRIPTIONAL REGULATORS IN CELLULAR STRESS . 28 3.1. The heat shock response . 28 3.2. Heat shock proteins . 28 3.3. The family of HSFs . 29 3.3.1. Functional domains of HSFs . 31 The DNA-binding domain . 31 Trimerization and interaction with the heat shock element . 32 The Hsp70 gene as a model for stress-induced transcription . 32 3.3.2. HSF1 . 33 Activation and regulation of HSF1 . 33 Post-translational modifications of HSF1 . 34 In search of the stress sensing mechanism of the heat shock response . 35 3.3.3. HSF2 . 36 Regulation of HSF2 . 37 Is there a role for HSF2 in the heat shock response? 38 3.4. Nuclear stress bodies . 39 4. ROLES OF HSF1 AND HSF2 BEYOND THE HEAT SHOCK RESPONSE . 41 4.1. HSF1 as a developmental factor . 41 4.2. HSF1 in aging and cancer . 42 4.3. HSF2 in developmental processes . 43 4.3.1. HSF2 in spermatogenesis . 44 Basic principles of spermatogenesis . 44 Hsf2 knockout phenotype in the testis . 46 HSF2 as a regulator of gene expression in spermatogenesis . 46 OUTLINE OF THE STUDY . 48 EXPERIMENTAL PROCEDURES . 49 1. Mice (II-III) . 49 2. Cell culture and experimental treatments (I-III) . 49 3. Plasmid construction (I-III) . 49 4. Transfection (I-III) . 50 5. Rna interference (RNAi) (I-II) . 50 6. Western blot analysis (I-III) . 51 7. Biotin-mediated oligonucleotide pulldown assay (I) . 51 8. Chromatin immunoprecipitation (ChIP) (I-II) . 52 9. Generation of recombinant proteins (II) . 53 10. Electrophoretic mobility shift assay (EMSA) (I-II) . 53 11. Immunoprecipitation (IP) (II) . 53 12. Immunofluorescence (II-III) . 54 13. Two-photon fluorescence lifetime imaging microscopy (FLIM) (II) . 55 14. Fluorescence-activated cell sorting-based fluorescence resonance energy transfer (FACS-based FRET) (II) . 55 15. Structural modeling (II) . 55 16. Semiquantitative reverse transcription (RT)-PCR and quantitative real-time RT-PCR (I-III) . 56 17. miRNA quantitative real-time RT-PCR (III) . 58 18. Luciferase assay (III) . 58 19. Transfection of germ cells in intact seminiferous tubules (T-GIST) (III) . 58 20. Squash preparations (III) . 59 21. Fluorescence-activated cell sorting (FACS) of transfected germ cells (III) . 59 22. In situ hybridization (III). 59 RESULTS AND DISCUSSION . 60 1. HSF2 AS A TRANSCRIPTIONAL REGULATOR IN THE HEAT SHOCK RESPONSE (I) . 60 1.1. Both HSF1 and HSF2 bind the Hsp70 promoter . 60 1.2. Recruitment of HSF2 to the Hsp70 promoter is HSF1-dependent . 61 1.3. HSF2 modulates the inducible expression of Hsps . 63 2. HETEROTRIMERIZATION BETWEEN HSF1 AND HSF2 (II) . 65 2.1. Translocation of HSF2 to the nSBs is HSF1-dependent . 65 2.2. Molecular modeling of heterotrimerization between HSF1 and HSF2 . 65 2.3. Heterotrimers of DNA-bound HSF1 and HSF2 . 66 2.4. Heterotrimerization as a regulatory step in HSF activity . 67 3. HSF2 INTERACTS WITH HSF1 IN STRESS AND DEVELOPMENT (I, II) . 69 3.1. HSF2 acts in concert with HSF1 during the heat shock response . 69 3.2. Putative HSF1/HSF2 interplay in development and consequences thereof . 70 4. REGULATION OF HSF2 BY miR-18-MEDIATED REPRESSION (III) . 71 4.1. Identification of the miR-18 binding site on Hsf2 . 72 4.2. Endogenous miR-18 targets Hsf2 . 72 4.3. Mutually exclusive expression of HSF2 and miR-18 in spermatogenesis . 73 4.4. miR-18 inhibition in spermatocytes affects HSF2 levels and target genes . 74 4.5. Aspects on miR-18-mediated regulation of HSF2 in spermatogenesis and beyond . 76 5.