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The Role of Sumoylation in Early Development of Xenopus Laevis And THE ROLE OF SUMOYLATION IN EARLY DEVELOPMENT OF XENOPUS LAEVIS AND REGULATION OF 5S RIBOSOMAL RNA GENES A Dissertation Submitted to the Graduate School of the University of Notre Dame in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Michelle M. Bertke Paul W. Huber, Director Graduate Program in Chemistry and Biochemistry Notre Dame, Indiana April 2014 THE ROLE OF SUMOYLATION IN EARLY DEVELOPMENT OF XENOPUS LAEVIS AND REGULATION OF 5S RIBOSOMAL RNA GENES Abstract by Michelle M. Bertke The 5S rRNA gene-specific transcription factor, TFIIIA, interacts with the SUMO E3 ligase, PIAS2b, and with one of its targets, the transcriptional corepressor XCtBP. PIAS2b and XCtBP are present on the oocyte, but not somatic, 5S rRNA genes up through the gastrula-neurula transition, as is a limiting amount of TFIIIA. Histone H3 methylation, coincident with the binding of XCtBP, also occurs exclusively on the oocyte genes. Immunohistochemical staining of embryos confirms occupancy of some fraction of the oocyte genes by TFIIIA that become positioned at the nuclear periphery shortly after the midblastula transition. SUMOylation can be inhibited through injection of mRNA encoding the adenovirus protein Gam1, which decreases the levels of the E1 activating enzyme by triggering its proteolytic degradation. Gam1-induced decrease in SUMOylation activity relieves repression of the oocyte 5S rRNA genes and is correlated with a decrease in methylation of H3K9 and H3K27. These results reveal a novel Michelle M. Bertke function for TFIIIA as a negative regulator that recruits histone modification activity, through the CtBP repressor complex, exclusively to the oocyte 5S rRNA genes, resulting in their terminal repression. SUMOylation deficient embryos also exhibit a range of important developmental defects including failure of the blastopore and neural tube to close, shortened axis, fused eyes, and perturbed heart development. Embryos injected with Gam1 mRNA or water (control) were taken for microarray analysis at three developmental time points: early gastrula, late gastrula, and early neurula. A bioinformatics analysis of this data was conducted using the MetaCore® suite of programs, BiNGO, DAVID, and the Gene Ontology database. Functional enrichment analysis of the differentially expressed genes demonstrates that SUMOylation regulates the expression of genes that span several different biological processes during early embryogenesis. Bioinformatics analysis provides evidence that, in some cases, SUMOylation generates two pools of a given transcription factor that control different subsets of genes. Although SUMOylation impacts a large variety of processes, certain signaling pathways appear to be particularly sensitive to the loss of this modification and can account for the observed phenotypes. Pathways enriched for differentially expressed genes were identified using the extensive MetaCore® database and include; non-canonical Wnt signaling and regulation of cytoskeleton remodeling (shortened axis and open blastopore), regulation by Yin Yang 1 (heart defects), Twist/Snail regulation of the epithelial to mesenchymal transition (open blastopore and neural tube), and Ets-1 regulation of transcription factors E2F1/E2F4 (heart defects and open blastopore). To my husband Jeffery for believing in me particularly when I did not. ii CONTENTS Figures .................................................................................................................................i v Tables .................................................................................................................................. vi Acknowledgments............................................................................................................. viii Introduction ........................................................................................................................ 1 Materials and Methods ..................................................................................................... 20 Results: Regulation of the 5S Ribosomal RNA Genes ...................................................... 47 Discussion: Regulation of the 5S Ribosomal RNA Genes ................................................. 68 Results and Discussion: Role of SUMOylation in Early Development of Xenopus laevis. 77 Appendix A ...................................................................................................................... 190 References ...................................................................................................................... 293 iii FIGURES Figure 1. An internal view of the early stages of Xenopus laevis development ................. 7 Figure 2. Conjugation of SUMO protein to a target. ........................................................ 15 Figure 3. Procedure for Keller sandwich explants. ........................................................... 36 Figure 4. Diagram of nucleus isolation for ChIP assays. ................................................... 39 Figure 5. Depletion of SUMOylation activity prevents repression of oocyte 5S rRNA genes. .................................................................................................................... 49 Figure 6. TFIIIA levels in Gam1-injected embryos. ........................................................... 51 Figure 7. Expression of Gam1 in embryos eliminates E1 enzyme activity. ...................... 53 Figure 8. SUMOylation of TFIIIA. ..................................................................................... 56 Figure 9. Occupancy of the 5S rRNA genes during early development. ........................... 61 Figure 10. Localization of TFIIIA to the nuclear periphery. ............................................... 66 Figure 11. Developmental defects of Gam1-injected embryos. ....................................... 81 Figure 12. Volcano plots and comparative histogram of genes measured on microarray.87 Figure 13. Comparison of biological replicates from Gam1 and H2O injected embryos. 91 Figure 14. Comparison of microarray and qRT-PCR data ................................................. 96 Figure 15. Functional enrichment analysis. .................................................................... 138 Figure 16. Heat maps of differentially expressed genes for three developmental time points. ................................................................................................................. 141 Figure 17. Syn-expression cluster analysis of differentially expressed genes. ............... 147 iv Figure 18. SUMO regulation of Wnt pathway leading to defects in cytoskeleton remodeling.. ........................................................................................................ 156 Figure 19. SUMO regulation of Twist/Snail which controls the epithelial to mesenchymal transition. ............................................................................................................ 169 Figure 20. SUMO regulation of Ets-1 and control of gastrulation and heart development. ............................................................................................................................. 175 Figure 21. Gene expression levels (log10) from microarray data compared to previously measured mRNA levels for genes regulated by Ets-1. ........................................ 182 Figure 22. Gam1 disrupts convergence and extension in Keller sandwich explants. ..... 187 v TABLES Table 1. Developmental stages of X. laevis from early blastula to late neurula. .............. 5 Table 2. Forward and reverse primers used for qRT-PCR analysis ................................... 29 Table 3. Top 30 transcription factors which regulate the largest number of differentially expressed genes at early gastrula along with their associated gene ontology processes............................................................................................................. 102 Table 4. Top 30 transcription factors which regulate the largest number of differentially expressed genes at late gastrula along with their associated gene ontology processes............................................................................................................. 107 Table 5. Top 30 transcription factors which regulate the largest number of differentially expressed genes at early neurula along with their associated gene ontology processes............................................................................................................. 113 Table 6. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at early gastrula along with their associated gene ontology processes. ............................................................................................ 118 Table 7. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at late gastrula along with their associated gene ontology processes. ............................................................................................ 122 Table 8. Top 30 transcription factors which regulate the largest number of non- differentially expressed genes at early neurula along with their associated gene ontology processes. ............................................................................................ 126 Table 9. Compiled list of transcription factors, identified to regulate differentially expressed genes from all three time points, analyzed for potential
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