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Genetic Analysis of Vprbp in Mice Sarah C. Jackson a Dissertation

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Genetic Analysis of Vprbp in Mice Sarah C. Jackson a Dissertation Genetic analysis of Vprbp in mice Sarah C. Jackson A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics. Chapel Hill 2011 Approved by: Yue Xiong, Ph.D. Jeanette Gowen Cook, Ph.D. William F. Marzluff, Ph.D. Robert J. Duronio, Ph.D. Norman E. Sharpless, M.D. ABSTRACT SARAH C. JACKSON: Genetic analysis of Vprbp in mice (Under the direction of Dr. Yue Xiong) Ubiquitin-mediated protein degradation is a critical mechanism for regulating many cellular functions. Substrate proteins are specified through the activity of an E3 ubiquitin ligase. Cullin proteins serve as scaffolds to assemble hundreds of distinct multi-subunit E3 ubiquitin ligase complexes. My research focused on CUL4-based complexes, which use linker protein DDB1 to bind one of approximately 100 putative substrate recruiting proteins. Specifically, I investigated HIV1 viral protein R binding protein (VprBP), a WD40 repeat-containing protein which binds to CUL4-DDB1 and is predicted to function as a substrate recognition subunit. Functional studies of VprBP remain limited; there are currently no known substrates for CUL4-DDB1-VprBP and Vprbp knockout mice are early embryonic lethal. In my work, I first tested a hypothesis that VprBP functions in epigenetic modification of histones and intriguingly observed a possible role in histone H4 ubiquitylation. My subsequent work focused on understanding the function and mechanism of Vprbp by analyzing conditional mouse embryonic fibroblasts (MEFs) and mice. I discovered that VprBP is required for MEF proliferation and paradoxically that high levels of VprBP protein are associated with quiescent cells. Finally, by crossing conditional Vprbp mice with transgenic Ubiquitin C promoter driven Cre-ERT2 + mice, I was able to temporally control Vprbp disruption in an unbiased manner to explore phenotypes beyond embryonic lethality This work uncovered a role for Vprbp in the proliferation and survival of lymphocytes in mice. ii ACKNOWLEDGEMENTS I would like to thank Yue, members of the Xiong lab, my committee members and my family for providing me with support and inspiration during my journey iii TABLE OF CONTENTS LIST OF TABLES..……………………………………………………………………………..vii LIST OF FIGURES………..……………………………………………………………………viii LIST OF ABBREVIATIONS.…………………………………………………………………….x Chapter I. INTRODUCTION ................................................................................................. 1 Ubiquitin-mediated protein degradation ....................................................... 2 Cullin Family Ubiquitin Ligases ................................................................... 3 Regulation of CRL Complexes .................................................................... 6 CRL4 complexes ........................................................................................ 8 Genetics analysis of CUL4 and DDB1 ....................................................... 10 Cullin 4 ubiquitin ligase complexes in DNA replication & repair ................. 16 Viral Hijacking of DDB1-CUL4 .................................................................. 20 HIV1 Vpr Binding Protein, VprBP .............................................................. 21 II. BIOCHEMICAL ANALYSIS OF VPRBP IN HISTONE MODIFICATION ........... 26 Summary …..…………………………………………………………….……...27 Background ............................................................................................ 28 Experimental Procedures ...................................................................... 31 Cell culture and plasmids .................................................................... 31 Cell transfection and RNAi .................................................................. 31 Mutagenesis ........................................................................................ 32 Cellular lysis, immunoprecipitation and western blot analysis .............. 32 iv Cell Cycle Synchronization and Flow Cytometry ................................. 33 DNA Damage Induction ....................................................................... 33 Results …..………………………………………………………………………34 VprBP may regulate global levels of histone ubiquitylation .................. 34 VprBP promotes H4 modification, but inhibits H1.1 modification .......... 34 H4 modification is potentially dependent on cell cycle phase . .............. 39 H4 modification is not induced by DNA damage .................................. 39 VprBP does not bind core histones ..................................................... 43 Discussion.. …………………………………………………………………….47 III. ANALYSIS OF VPRBP DISRUPTION IN MOUSE EMBRYONIC FIBROBLASTS ............................................................................................... 49 Summary ……...…………………………………………………………………50 Background ............................................................................................. 51 Experimental Procedures ....................................................................... 53 Animal maintenance and generation of mouse embryonic fibroblasts . 53 Genotyping .......................................................................................... 53 Cell Culture ......................................................................................... 54 Cell lysis and western blot analysis ..................................................... 54 Flow Cytometry ................................................................................... 54 Results ……..……………………………………………………………………56 Characterizing Vprbp ∆ ......................................................................... 56 Vprbp ∆ is a hypomorphic allele that mimics loss of function ................. 58 Evidence for VprBP accumulation in quiescence ................................ 62 Discussion …. .......................................................................................... 70 IV. INDUCIBLE DISRUPTION OF VPRBP IN MOUSE IMPAIRS T-CELL DEVELOPMENT, PROLIFERATION AND SURVIVAL ................................... 73 Summary …...……………………………………………………………………74 v Background ............................................................................................. 75 Experimental Procedures ....................................................................... 78 Mice …………………………………………………………………………..78 Genotyping .......................................................................................... 78 Immunohistochemistry and Immunoblotting ........................................ 79 Flow Cytometry ................................................................................... 80 Cell Culture ......................................................................................... 80 qPCR …………………………………………………………………………81 Results ………………………………………………..…………………………82 Inducible disruption of Vprbp results in marked thymic atrophy ........... 82 Defects in T-lymphocyte development following inducible disruption of Vprbp .............................................................................. 84 Disruption of Vprbp decreased proliferation and induced apoptosis in thymus ............................................................................. 86 Inducible disruption of Vprbp disrupts B-cell development. .................. 87 Mature T-cells with disrupted Vprbp fail to proliferate in response to activation ......................................................................................... 90 Loss of Ddb1 disrupts B- and T-cell development . .............................. 94 Discussion ............................................................................................... 96 V. CONCLUSIONS AND PERSPECTIVES ......................................................... 99 Does VprBP have CRL4-independent functions? .................................... 100 VprBP likely targets a diverse set of substrates ...................................... 102 Identification of bona fide CRL4 VprBP substrates ...................................... 104 Future Genetic Studies ........................................................................... 107 REFERENCES ................................................................................................... 115 vi LIST OF TABLES Table Page 1.1 CRL4 Substrates…………………...………………………………………… 18 vii LIST OF FIGURES Figure Page 1.1 Cullin-RING ubiquitin ligases……….………………………………………….. 4 1.2 Schematic comparison of CUL4 proteins and mouse Cul4a mutants……… 12 1.3 Schematic of VprBP……………………………………………………………… 23 2.1 VprBP regulates global levels of H4 ubiquitylation……………………………. 35 2.2 VprBP inhibits H1.1 ubiquitylation………………………………………………. 36 2.3 H4 modification is potentially cell cycle dependent …….…………………….. 41 2.4 H4 modification is not induced by DNA damage …….……………………...… 42 2.5 Multiple sites affect H4 modification……………………..……………………… 45 2.6 VprBP does not stably bind core histone H4 ………………………………….. 46 3.1 Vprbp genomic locus and alleles………………………………………………... 57 3.2 Vprbp ∆ is a hypomorphic allele………………………………………………….. 59 3.3 Vprbp ∆ mimics loss of function………………………………………………...… 60 3.4 VprBP accumulates in response to serum starvation in MEFs……………… 63 3.5 VprBP accumulates in highly confluent MEFs……………..…………………... 65 3.6 VprBP accumulation is cell type dependent……………………………….…… 66 3.7 Vprbp is inefficient following serum starvation………………………...........…. 69 4.1 Inducible disruption of Vprbp results in marked thymic atrophy...…….…...…
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