The Role of Sumoylation in Early Development of Xenopus laevis and Regulation of 5S Ribosomal RNA Genes

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 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(R) 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(R) 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).