Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2007 Death by Frustration: How Defects in Cell Differentiation Trigger Apoptosis Ann H. Tang Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Tang, Ann H., "Death by Frustration: How Defects in Cell Differentiation Trigger Apoptosis" (2007). Student Theses and Dissertations. Paper 187. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. DEATH BY FRUSTRATION: HOW DEFECTS IN CELL DIFFERENTIATION TRIGGER APOPTOSIS A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for The degree of Doctor of Philosophy by Ann H. Tang June 2007 Title Page Copyright Page © Copyright by Ann H. Tang 2007 DEATH BY FRUSTRATION: HOW DEFECTS IN CELL DIFFERENTIATION TRIGGER APOPTOSIS Ann H. Tang, Ph.D. The Rockefeller University 2007 Apoptosis plays a crucial role in the growth, development, and homeostasis of multicellular organisms by removing excess cells and sculpting tissues. The implications of its misregulation in diseases such as cancer, neurodegeneration, and autoimmunity bespeak of its importance. A family of proteases called caspases brings about the orderly deconstruction of a cell through an evolutionarily-conserved process. A bewildering number of molecules have been implicated to either directly or indirectly regulate these enzymes. Since the net outcome between inhibitory and activational mechanisms ultimately determines a cell’s fate, caspases and their regulation still continue to be subjects of intense study. Studies with dcp-1 described here have clarified its role as a Drosophila effector caspase. From the original descriptions of its somatic phenotypes, a possible role for dcp-1 in cell proliferation was initially explored, but these phenotypes were eventually found to be associated with a disruption in another gene encoded in the same locus. RNAi studies with dcp-1 and drice, another effector caspase, have provided in vivo confirmation of their epistatic relationship to rpr, hid, and grim and have revealed that they may work synergistically. rpr indirectly activates caspases by derepressing their inhibitors, and many proapoptotic signals trigger its transcriptional upregulation. An analysis of its promoter described here has furthered the understanding of how developmental mutations cue rpr transcription. As a consequence of signaling resulting from developmentally aberrant cells, rpr is transcriptionally activated via different elements in its promoter region, rather than through a single one, suggesting the role of different pathways and hence different transcription factors. A yeast 1-hybrid assay performed with a narrow region that can mediate upregulation in crb and srp3 backgrounds has led to the identification of a transcriptional modulator of rpr, p8. Its mammalian homolog has been implicated in stress response and apoptosis. The induced RNA and protein levels of p8, the RNA in situ pattern, and a reduction of reporter signal in p8 mutants in crb and srp3 backgrounds support a role for p8 in transducing a pro-apoptotic response resulting from developmental mutants to the activation of rpr transcription. Dedication Dedicated to my loving parents. iii Acknowledgments First and foremost, I thank Hermann Steller for the privilege of working with him and for his support, mentorship, enthusiasm, and patience. I am grateful to him for allowing me the time, space, and freedom to learn to independently solve problems I encountered along the way. I especially appreciate that he assembles a lab with talented and dedicated people, encourages good rapport amongst ourselves, and sets a tone of high integrity for research. I thank the members of my committee, Shai Shaham and Michael Young, for their encouragement, comments, and interest. In addition, I thank Michael Young for having welcomed me into his lab for my first rotation at the beginning of my graduate career. And I thank my outside committee member, Kristin White, for her time and insight. I thank the current and former members of the Steller Lab for their help, friendship, and invaluable discussions. I especially want to thank Eli Arama and Joe Rodriguez for their generosity, moral support, and entertaining discussions in all matters scientific and otherwise. I am grateful to them and Gabrielle Rieckhof for helping me through the final stretch. I thank Steve Anderson for always believing in me, encouraging me, and thinking of me. Finally, I thank my family and friends for their unwavering love and support. iv Table of Contents Title Page i Copyright ii Dedication iii Acknowledgments iv Table of Contents v List of Figures viii List of Tables x Abbreviations xi 1 Introduction to Programmed Cell Death 1 1.1 Prologue 1 1.2 Relevance 2 1.3 The core apoptotic pathway 4 1.4 Studying apoptosis with D. melanogaster 16 1.5 Drosophila caspases 17 1.6 Drosophila RHG genes 20 1.7 reaper and its transcriptional regulation 24 1.8 Transcriptional regulation of apoptotic genes 26 2 Characterization of dcp-1 31 2.1 Introduction 31 2.2 Materials and methods 35 2.2.1 Drosophila stocks 35 2.2.2 Generation of transgenic flies 35 2.2.3 Generation of αDCP-1 antibody 36 2.2.4 X-gal staining of imaginal discs ` 36 2.2.5 Antibody staining of imaginal discs 37 2.2.6 BrdU incorporation 38 2.3 Results 38 2.3.1 Analysis of small imaginal disc phenotype 38 2.3.2 Determination of gene responsible for phenotypes 43 v 2.3.3 Epistatic relationship of dcp-1 and drice to rpr, hid, and grim 55 2.4 Conclusions 58 3 Promoter analysis of reaper to identify a factor that mediates death from improper differentiation 63 3.1 Introduction 63 3.2 Materials and methods 66 3.2.1 Drosophila stocks 66 3.2.2 Generation of transgenic flies 66 3.2.3 Antibody staining of embryos 69 3.2.4 Antibody staining of imaginal discs 70 3.2.5 X-gal staining of embryos 70 3.2.6 TUNEL staining of embryos 71 3.2.7 RNA in situ hybridization of embryos 71 3.2.8 Yeast 1-hybrid assay 73 3.2.9 Generation of αP8 antibody 74 3.2.10 Affinity purification of αP8 antibody 75 3.3 Identification of reaper cis-regulatory sequences responding to defects in cell differentiation 76 3.3.1 Background 76 3.3.2 Identification of response elements in ~600 bp region 79 3.3.3 Identification of response elements in at5, a 175 bp reaper promoter fragment 83 3.3.4 Further truncations of 175 bp at5 region 87 3.3.5 Role of at5 reaper response elements in other developmental mutants 91 3.4 Identification of p8 as a putative transcriptional activator of reaper 96 3.4.1 Yeast 1-hybrid assay to screen for putative factors 96 3.4.2 Evaluation of p8 101 3.5 Conclusion 116 vi 3.5.1 Integration of different pro-apoptotic signals at the reaper promoter in response to developmental frustration 116 3.5.2 p8 as a modulator of reaper transcription 119 4 Implications of the current study and prospects for future Research 121 4.1 Introduction 121 4.2 Further analysis of the reaper promoter 121 4.3 Further characterization of p8 123 vii List of Figures Figure 1.1: The core apoptotic machinery 14 Figure 2.1: Mutant phenotypes associated with l(2)1862 and l(2)2132 32 Figure 2.2: Northern analysis for dcp-1 and pita trancripts 34 Figure 2.3: Evaluation of patterning in imaginal discs of l(2)2132 3rd instar larvae 37 Figure 2.4: Cell autonomous requirement for gene disrupted by l(2)2132 42 Figure 2.5: Absence of cell cycle defect in l(2)2132 clones 44 Figure 2.6: Absence of effect of inhibition of activated caspases on clonal size 47 Figure 2.7: Absence of effect of reduction of DCP-1 by DN-DCP-1 on clonal size 48 Figure 2.8: Absence of effect of reduction of DCP-1 by dcp-1-RNAi on clonal size 50 Figure 2.9: Scheme for isolation of additional l(2)2132 alleles 51 Figure 2.10: Mutant C7.8 from genetic screen 53 Figure 2.11: Absence of effect of overexpression of pita on clonal size 57 Figure 2.12: Suppression of GMR-rpr by reduction of effector caspases 60 Figure 2.13: Suppression of GMR-hid by reduction of effector caspases 61 Figure 2.14: Suppression of GMR-grim by reduction of effector caspases 62 Figure 3.1: Schematic of death by frustration 65 Figure 3.2: Upregulation of rpr-LacZ lines in crb and srp3 mutants 78 Figure 3.3: Analysis of ~600bp promoter fragments from a 4 kb region 81 Figure 3.4: Analysis of promoter segments truncated from 4s1 region 85 Figure 3.5: Analysis of promoter segments truncated from at5 88 Figure 3.6: Subdivision of fragment at5a 90 Figure 3.7: Determination of the extent of the requirement of the rpr promoter sequences for a transcriptional response in bcd and dl mutants 92 Figure 3.8: Subdivision of 4kb promoter region 94 viii Figure 3.9: Reporter upregulation in ph but not dl or bcd 95 Figure 3.10: Aligment of P8 protein sequences between Drosophila and mammalian species 100 Figure 3.11: RNA in situ hybridization of p8 transcript in embryos 102 Figure 3.12: Loss-of-function p-alleles of p8 107 Figure 3.13: Decrease of β-galactosidase in p8 mutants 109 Figure 3.14: Absence of effect on rpr endogenous levels and on apoptosis in p8 mutants 110 Figure 3.15: αP8 antibody detects overexpressed but not endogenous P8 111 Figure 3.16: Confirmation of p8 p-alleles as loss-of-function 112 Figure 3.17: Increase