Abstract
The ubiquitin (Ub)-dependent N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A substrate with destabilizing N-terminal residue is recognized by a family of mammalian E3 ubiquitin ligases of the N-end rule pathway, including UBR1 and UBR2. However, little is known about their roles in biological processes. The aim of this dissertation is to understand the physiological functions and underlying molecular mechanisms of the E3 ubiquitin ligases in the N-end rule pathway. First, I find that UBR2, a recognition E3 component of the N-end rule pathway, localizes to the meiotic chromatin, where it mediates the transcriptional silencing through histone ubiquitylation in a spatiotemporal manner. UBR2-lacking spermatocytes show impaired global ubiquitylation, including ubiquitylation of histone H2A, a histone modification often associated with transcriptional inactivation. HR6B, an E2 conjugating enzyme of the N-end rule pathway, interacts with UBR2 as an E2-E3 complex and cooperatively mediate H2A monoubiquitylation in vitro. Impaired H2A ubiquitylation in UBR2-deficient spermatocytes correlates to defects in transcriptional silencing. Furthermore, I provide evidences that UBR2 is involved in DNA damage response (DDR) pathway through protein ubiquitylation presumably to maintain genome integrity. UBR2 is exclusively associated with non-heterochromatin throughout nucleus and responds to genotoxic stress via post-translational modification. Ubiquitylation induced by DNA damage are significantly impaired in UBR2-deficient cells. UBR2-/- mouse embryonic fibroblasts (MEFs) show increased vulnerability to the genotoxic agents and abnormality of chromosomes. Moreover, I show that divergent and cooperative functions of the E3 ligases of N-end rule pathway. UBR1 and UBR2 are 46% identical, and appear to be indistinguishable in their recognition of N degrons, yet show different physiological implications in mutant mice. UBR1-/-UBR2-/- embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects include reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D type cyclins and Notch1 is also altered in UBR1-/-UBR2-/- embryos. Overall, my dissertation suggests that the E3 Ub-ligases of the N-end rule pathway are required in meiosis, DDR pathway, and embryogenesis.
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