SUMOylation is enriched in the nuclear matrix and required for chromosome segregation.

As an important posttranslational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or KO of SUMO-conjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 KO cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes.

Control of SOX2 protein stability and tumorigenic activity by E3 ligase CHIP in esophageal cancer cells.

SOX2 is highly expressed and controls tumor initiation and cancer stem cell function in various squamous cell carcinomas including esophageal squamous cancer. However, the molecular mechanism leading to SOX2 overexpression in cancer is incompletely understood. Here, we identified CHIP, a chaperone-associated ubiquitin E3 ligase, as a novel negative regulator of SOX2 protein stability and tumorigenic activity in esophageal squamous carcinoma cells. We showed that CHIP interacted with SOX2 primarily via chaperone HSP70, together they catalyzed SOX2 ubiquitination and degradation via proteasome. In contrast, HSP90 promoted SOX2 stability and inhibition of HSP90 activity induced SOX2 ubiquitination and degradation. Notably, unlike the case in normal esophageal tissues where CHIP was detected in both the cytoplasm and nucleus, CHIP in clinical esophageal tumor specimens was predominantly localized in the cytoplasm. Consistent with this observation, we observed increased expression of exportin-1/CRM-1 in clinical esophageal tumor specimens. We further demonstrated that CHIP catalyzed SOX2 ubiquitination and degradation primarily in the nuclear compartment. Taken together, our study has identified CHIP as a key suppressor of SOX2 protein stability and tumorigenic activity and revealed CHIP nuclear exclusion as a potential mechanism for aberrant SOX2 overexpression in esophageal cancer. Our study also suggests HSP90 inhibitors as potential therapeutic agents for SOX2-positive cancers.

MYC promotes global transcription in part by controlling P-TEFb complex formation via DNA-binding independent inhibition of CDK9 SUMOylation.

MYC is an oncogenic transcription factor with a novel role in enhancing global transcription when overexpressed. However, how MYC promotes global transcription remains controversial. Here, we used a series of MYC mutants to dissect the molecular basis for MYC-driven global transcription. We found that MYC mutants deficient in DNA binding or known transcriptional activation activities can still promote global transcription and enhance serine 2 phosphorylation (Ser2P) of the RNA polymerase (Pol) II C-terminal domain (CTD), a hallmark of active elongating RNA Pol II. Two distinct regions within MYC can promote global transcription and Ser2P of Pol II CTD. The ability of various MYC mutants to promote global transcription and Ser2P correlates with their ability to suppress CDK9 SUMOylation and enhance positive transcription elongation factor b (P-TEFb) complex formation. We showed that MYC suppresses CDK9 SUMOylation by inhibiting the interaction between CDK9 and SUMO enzymes including UBC9 and PIAS1. Furthermore, MYC's activity in enhancing global transcription positively contributes to its activity in promoting cell proliferation and transformation. Together, our study demonstrates that MYC promotes global transcription, at least in part, by promoting the formation of the active P-TEFb complex via a sequence-specific DNA-binding activity-independent manner.

Nuclear UHRF1 is a gate-keeper of cellular AMPK activity and function.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis. Although much has been learned on how low energy status and glucose starvation activate AMPK, how AMPK activity is properly controlled in vivo is still poorly understood. Here we report that UHRF1, an epigenetic regulator highly expressed in proliferating and cancer cells, interacts with AMPK and serves to suppress AMPK activity under both basal and stressed conditions. As a nuclear protein, UHRF1 promotes AMPK nuclear retention and strongly suppresses nuclear AMPK activity toward substrates H2B and EZH2. Importantly, we demonstrate that UHRF1 also robustly inhibits AMPK activity in the cytoplasm compartment, most likely as a consequence of AMPK nucleocytoplasmic shuttling. Mechanistically, we found that UHRF1 has no obvious effect on AMPK activation by upstream kinases LKB1 and CAMKK2 but inhibits AMPK activity by acting as a bridging factor targeting phosphatase PP2A to dephosphorylate AMPK. Hepatic overexpression of UHRF1 showed profound effects on glucose and lipid metabolism in wild-type mice but not in those with the liver-specific knockout of AMPKα1/α2, whereas knockdown of UHRF1 in adipose tissue led to AMPK activation and reduced sizes of adipocytes and lipogenic activity, highlighting the physiological significance of this regulation in glucose and lipid metabolism. Thus, our study identifies UHRF1 as a novel AMPK gate-keeper with critical roles in cellular metabolism.

Linking nuclear matrix-localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z.

As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

USP7 negatively controls global DNA methylation by attenuating ubiquitinated histone-dependent DNMT1 recruitment.

Previous studies have implicated an essential role for UHRF1-mediated histone H3 ubiquitination in recruiting DNMT1 to replication sites for DNA maintenance methylation during S phase of the cell cycle. However, the regulatory mechanism on UHRF1-mediated histone ubiquitination is not clear. Here we present evidence that UHRF1 and USP7 oppositely control ubiquitination of histones H3 and H2B in S phase of the cell cycle and that DNMT1 binds both ubiquitinated H3 and H2B. USP7 knockout markedly increased the levels of ubiquitinated H3 and H2B in S phase, the association of DNMT1 with replication sites and importantly, led to a progressive increase of global DNA methylation shown with increased cell passages. Using DNMT3A/DNMT3B/USP7 triple knockout cells and various DNA methylation analyses, we demonstrated that USP7 knockout led to an overall elevation of DNA methylation levels. Mechanistic study demonstrated that USP7 suppresses DNMT1 recruitment and DNA methylation through its deubiquitinase activity and the interaction with DNMT1. Altogether our study provides evidence that USP7 is a negative regulator of global DNA methylation and that USP7 protects the genome from excessive DNA methylation by attenuating histone ubiquitination-dependent DNMT1 recruitment.

SET8 prevents excessive DNA methylation by methylation-mediated degradation of UHRF1 and DNMT1.

Faithful inheritance of DNA methylation across cell division requires DNMT1 and its accessory factor UHRF1. However, how this axis is regulated to ensure DNA methylation homeostasis remains poorly understood. Here we show that SET8, a cell-cycle-regulated protein methyltransferase, controls protein stability of both UHRF1 and DNMT1 through methylation-mediated, ubiquitin-dependent degradation and consequently prevents excessive DNA methylation. SET8 methylates UHRF1 at lysine 385 and this modification leads to ubiquitination and degradation of UHRF1. In contrast, LSD1 stabilizes both UHRF1 and DNMT1 by demethylation. Importantly, SET8 and LSD1 oppositely regulate global DNA methylation and do so most likely through regulating the level of UHRF1 than DNMT1. Finally, we show that UHRF1 downregulation in G2/M by SET8 has a role in suppressing DNMT1-mediated methylation on post-replicated DNA. Altogether, our study reveals a novel role of SET8 in promoting DNA methylation homeostasis and identifies UHRF1 as the hub for tuning DNA methylation through dynamic protein methylation.