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Ubiquitination of histones provides an important mechanism regulating chromatin remodeling and gene expression. Recent studies have revealed ubiquitin ligases involved in histone ubiquitination, yet the responsible enzymes and the function of histone ubiquitination in spermatogenesis remain unclear. We have previously shown that mice lacking the ubiquitin ligase UBR2, one of the recognition E3 components of the N-end rule proteolytic pathway, are infertile associated with meiotic arrest at prophase I. We here show that UBR2 localizes to meiotic chromatin regions, including unsynapsed axial elements linked to chromatin inactivation, and mediates transcriptional silencing via the ubiquitination of histone H2A. UBR2 interacts with the ubiquitin conjugating enzyme HR6B and its substrate H2A and promotes the HR6B–H2A interaction and the HR6B-to-H2A transfer of ubiquitin. UBR2 and ubiquitinated H2A (uH2A) spatiotemporally mark meiotic chromatin regions subject to transcriptional silencing, and UBR2-deficient spermatocytes fail to induce the ubiquitination of H2A during meiosis. UBR2-deficient spermatocytes are profoundly impaired in chromosome-wide transcriptional silencing of genes linked to unsynapsed axes of the X and Y chromosomes. Our findings suggest that insufficiency in UBR2-dependent histone ubiquitination triggers a pachytene checkpoint system, providing a new insight into chromatin remodeling and gene expression regulation.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A subset of degradation signals recognized by the N-end rule pathway comprises the signals, called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified a family of at least four mammalian E3 ubiquitin ligases, including UBR1 and UBR2, that share the UBR box and recognize N-degrons. These E3 enzymes mediate the multifunctional N-end rule pathway, but their individual roles are just beginning to emerge. Mutations of UBR1 in humans are the cause of Johanson–Blizzard syndrome. UBR1 and UBR2 are 46% identical and appear to be indistinguishable in their recognition of N-degrons. UBR1 −/− mice are viable but have defects that include pancreatic insufficiency, similarly to UBR1 −/− human patients with Johanson–Blizzard syndrome. UBR2 −/− mice are inviable in some strain backgrounds and are defective in male meiosis. To examine functional relationships between UBR1 and UBR2, we constructed mouse strains lacking both of these E3s. We report here that UBR1 −/− UBR2 −/− embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects included reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D-type cyclins and Notch1 was also altered in UBR1 −/− UBR2 −/− embryos. We conclude that the functions of UBR1 and UBR2 are significantly divergent, in part because of differences in their expression patterns and possibly also because of differences in their recognition of protein substrates that contain degradation signals other than N-degrons.

The ATE1 -encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1 –/– embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo . We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1 –/– embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and β-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O 2 -ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.