Double homeobox 4 activates germline genes, endogenous retroelements and immune modulators: Implications for facioscapulohumeral muscular dystrophy Linda N. Geng A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2011 Stephen J. Tapscott, Chair Edith H. Wang Muneesh Tewari Program Authorized to Offer Degree: Molecular and Cellular Biology University of Washington Abstract Double homeobox 4 activates germline genes, endogenous retroelements and immune modulators: Implications for facioscapulohumeral muscular dystrophy Linda N. Geng Chair of Supervisory Committee: Professor Stephen J. Tapscott Department of Neurology Double homeobox 4 (DUX4) is a candidate disease gene for causing facioscapulohumeral dystrophy (FSHD), a condition characterized by progressive degeneration of specific skeletal muscle groups. While the genetic lesion associated with the disease has been known for decades, the molecular mechanism(s) leading to muscular dystrophy remained unclear. DUX4 was detected in FSHD muscle, but the abundance was extremely low and the downstream consequence of DUX4 in human muscle cells was not known. This work demonstrates that the low level of DUX4 expression in a population of FSHD muscle cells represents a relatively high expression of DUX4 mRNA and protein in a subset of cells at any given point in time. While the full length DUX4 induces toxicity in muscle cells, the shorter isoform without the C-terminal end, DUX4-s, exhibited no obvious detrimental effects on muscle cells. Genome-wide binding studies revealed that DUX4 protein binds to a DNA target sequence that contains two closely-spaced canonical homeodomain binding sequences TAAT in tandem. These binding sites are present in both unique regions of the genome as well as within LTRs of a family of endogenous retrotransposons called MaLR. DUX4-s can bind to the same sequences, but, interestingly, DUX4-s was unable to activate transcription in luciferase reporter assays and, in fact, could act as a dominant negative to inhibit full length DUX4’s activity. Microarray expression analysis showed that DUX4 can upregulate over a thousand genes involved in multiple processes, such as germ cell development, RNA splicing and immune modulation. These targets are indeed upregulated in cultured FSHD muscle cells as well as biopsies, providing further support for the causal role of DUX4 in FSHD. One of the DUX4 targets, DEFB103, not only suppresses the innate immune response to viral infection but also inhibits myogenic differentiation. Therefore, FSHD represents a disease where incomplete developmental silencing of a retrogene DUX4 results in inappropriate expression of its targets in skeletal muscle cells. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression. Table of Contents LIST OF FIGURES .................................................................................................................................. ii LIST OF TABLES ................................................................................................................................... iii Chapter 1: Introduction ....................................................................................................................... 1 Chapter 2: Immunodetection of double homeobox 4 ....................................................................... 7 Summary ........................................................................................................................................... 8 Introduction ...................................................................................................................................... 8 Results ............................................................................................................................................... 9 Discussion ........................................................................................................................................ 12 Materials and Methods ................................................................................................................... 13 Chapter 3: FSHD: incomplete suppression of a retrotransposed gene .......................................... 22 Summary ......................................................................................................................................... 23 Introduction .................................................................................................................................... 23 Results ............................................................................................................................................. 26 Discussion ........................................................................................................................................ 32 Materials and Methods ................................................................................................................... 35 Chapter 4: DUX4 induces germline genes in skeletal muscle of individuals with FSHD ............... 56 Summary ......................................................................................................................................... 57 Introduction .................................................................................................................................... 57 Results ............................................................................................................................................. 59 Discussion ........................................................................................................................................ 66 Materials and Methods ................................................................................................................... 69 Chapter 5: Discussion ...................................................................................................................... 163 LIST OF REFERENCES ...................................................................................................................... 172 i LIST OF FIGURES Figure # Page 2.1 Production of recombinant DUX4 antigen 17 2.2 Reactivity of monoclonal antibodies on western blot 18 2.3 Reactivity of monoclonal antibodies on 19 immunofluorescence 2.4 Effects of DUX4 in muscle cells 20 3.1 Expression of DUX4-fl and DUX4-s and D4Z4 in control 46 and FSHD cells 3.2 A small number of FSHD muscle cells express a relatively 47 large amount of DUX4 3.3 Expression of DUX4-fl in human tissues 48 3.4 Expression of DUX4 in the testis 49 3.5 Alternative exon and polyadenylation site usage in germ- 50 line and somatic tissues 3.6 Expression of DUX4-fl and DUX4-s in pluripotent stem 51 cells and differentiated tissues 4.1 DUX4-fl activates the expression of germline genes and 149 binds a double-homeobox motif 4.2 DUX4-fl activates transcription in vivo and DUX4-s can 150 interfere with its activity 4.3 DUX4 targets are normally expressed in human testis but 151 not in healthy skeletal muscle 4.4 DUX4 regulated genes normally expressed in the testis 152 are aberrantly expressed in FSHD muscle 4.5 DEFB103 inhibits innate immune response to viral 153 infection and inhibits muscle differentiation S4.1 RT-PCR validation of DUX4-fl target genes from 154 expression microarray S4.2 Antibody characterization 155 S4.3 DUX4 binding in repeat and non-repeat regions and 156 EMSA validation of DUX4 binding to ChIP-seq determined motifs S4.4 Global DUX4-fl binding is moderately associated with the 157 expression of its targets, but DUX4-fl can act as an enhancer at certain loci S4.5 DUX4-fl expression status in muscle samples and 158 inhibition with dominant negative DUX4-s ii LIST OF TABLES Table # Page 2.1 Properties of anti-DUX4 monoclonal antibodies 16 3.1 DUX4 mRNA expression in FSHD and control biopsies 42 3.2 DUX4 mRNA expression in FSHD and control cell lines 43 3.3 Haplotype identification of DUX4 mRNA in human testis 44 3.4 Diagnostic polymorphisms in exon 2 45 4.1 Representative genes induced by DUX4-fl 75 4.2 DUX4 highly activates gene families involved in germ cell 77 and early development S4.1 Expression array analysis of DUX4-fl and DUX4-s in 78 cultured human skeletal muscle S4.2 Gene Ontology analysis of genes up-regulated by DUX4-fl 127 S4.3 Gene Ontology analysis of genes down-regulated by 129 DUX4-fl S4.4 Gene Ontology analysis of genes up-regulated by DUX4-fl 131 eight-fold or more S4.5 Repeat families bound by DUX4 132 S4.6 Non-repeat element DUX4-fl binding sites associated 133 with expressed genes S4.7 DUX4-fl expression in FSHD and control muscle 136 S4.8 Genes induced by lenti-GFP and lenti-DUX4-s but poorly 137 induced by lenti-DUX4-fl S4.9 Genes differentially expressed in DEFB103-treated 147 versus control cultured muscle cells during differentiation iii ACKNOWLEDGMENTS I offer my sincerest gratitude to my supervisor Dr. Stephen Tapscott for his invaluable support, guidance and wisdom. I also wish to thank everyone in the Tapscott lab for being not only my scientific colleagues but also my friends and mentors. I would like to further extend my appreciation to collaborators at the Fred Hutchinson and elsewhere who have greatly aided in the progression of this work and to my doctoral thesis committee who have helped shape me as a scientist. I stand on the shoulder of giants. iv DEDICATION To my parents and O.S.P. v 1 Chapter 1: Introduction This chapter