SUMMARY Histone H2B monoubiquitination (H2Bub1) is centrally involved in gene regulation. The deubiquitination module (DUBm) of the SAGA complex is a major regulator of global H2Bub1 levels, and components of this DUBm are linked to both neurodegenerative diseases and cancer. Unexpectedly, we find that ablation of USP22, the enzymatic center of the DUBm, leads to a reduction, rather than an increase, in global H2bub1 levels. In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two new H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and which compete with USP22 for ATXN7L3 and ENY2 for activity. Like USP22, USP51 and USP27X are required for normal cell proliferation, and their depletion suppresses tumor growth. Our results reveal that ATXN7L3 and ENY2 orchestrate activities of multiple deubiquitinating enzymes and that imbalances in these activities likely potentiate human diseases including cancer.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex possesses acetyltransferase and deubiquitinase activities. Within this modular complex, Ataxin-7 anchors the deubiquitinase activity to the larger complex. Here we identified and characterized Drosophila Ataxin-7 and found that reduction of Ataxin-7 protein results in loss of components from the SAGA complex. In contrast to yeast, where loss of Ataxin-7 inactivates the deubiquitinase and results in increased H2B ubiquitination, loss of Ataxin-7 results in decreased H2B ubiquitination and H3K9 acetylation without affecting other histone marks. Interestingly, the effect on ubiquitination was conserved in human cells, suggesting a novel mechanism regulating histone deubiquitination in higher organisms. Consistent with this mechanism in vivo, we found that a recombinant deubiquitinase module is active in the absence of Ataxin-7 in vitro. When we examined the consequences of reduced Ataxin-7 in vivo, we found that flies exhibited pronounced neural and retinal degeneration, impaired movement, and early lethality.

A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. Yet, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for Epidermal Growth Factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGFR-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion.

Previous studies have demonstrated that the base excision repair enzyme thymine DNA glycosylase (TDG) mediates recruitment of histone acetyltransferases CREB-binding protein (CBP) and p300 to DNA, suggesting a plausible role for these factors in TDG-mediated repair. Furthermore, TDG was found to potentiate CBP/p300-dependent transcription and serve as a substrate for CBP/p300 acetylation. Here, we show that the small ubiquitin-like modifier 1 (SUMO-1) protein binding activity of TDG is essential for activation of CBP and localization to promyelocytic leukemia protein oncogenic domains (PODs). SUMO-1 binding is mediated by two distinct amino-and carboxy-terminal motifs (residues 144 to 148 and 319 to 322) that are negatively regulated by DNA binding via an amino-terminal hydrophilic region (residues 1 to 121). TDG is also posttranslationally modified by covalent conjugation of SUMO-1 (sumoylation) to lysine 341. Interestingly, we found that sumoylation of TDG blocks interaction with CBP and prevents TDG acetylation in vitro. Furthermore, sumoylation effectively abrogates intermolecular SUMO-1 binding and a sumoylation-deficient mutant accumulates in PODs, suggesting that sumoylation negatively regulates translocation to these nuclear structures. These findings suggest that TDG sumoylation promotes intramolecular interactions with amino-and carboxy-terminal SUMO-1 binding motifs that dramatically alter the biochemical properties and subcellular localization of TDG.

CpG dinucleotides are mutational hotspots associated with cancer and genetic diseases. Thymine DNA glycosylase (TDG) plays an integral role in CpG maintenance by excising mispaired thymine and uracil in a CpG context and also participates in transcriptional regulation via gene-specific CpG demethylation and functional interactions with the transcription machinery. Here, we report that protein kinase C α (PKCα) interacts with TDG and phosphorylates amino-terminal serine residues adjacent to lysines acetylated by CREB-binding protein (CBP) and p300 (CBP/p300). We establish that acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG. Remarkably, acetylation of the amino-terminal region abrogates high-affinity DNA binding and selectively prevents processing of G:T mispairs. In contrast, phosphorylation does not markedly alter DNA interactions, but may preserve G:T processing in vivo by preventing CBP-mediated acetylation. Mutational analysis suggests that the acetyl-acceptor lysines are not directly involved in contacting DNA, but may constitute a conformationally sensitive interface that modulates DNA interactions. These findings reveal opposing roles of CBP/p300 and PKCα in regulating the DNA repair functions of TDG and suggest that the interplay of these modifications in vivo may be critically important in the maintenance of CpG dinucleotides and epigenetic regulation.