Huadong Pei

FKBP51 Affects Cancer Cell Response to Chemotherapy by Negatively Regulating Akt

Akt is a central regulator of cell growth. Its activity can be negatively regulated by the phosphatase PHLPP that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1). However, how PHLPP is targeted to Akt is not clear. Here we show that FKBP51 (FK506-binding protein 51) acts as a scaffolding protein for Akt and PHLPP and promotes dephosphorylation of Akt. Furthermore, FKBP51 is downregulated in pancreatic cancer tissue samples and several cancer cell lines. Decreased FKBP51 expression in cancer cells results in hyperphosphorylation of Akt and decreased cell death following genotoxic stress. Overall, our findings identify FKBP51 as a negative regulator of the Akt pathway, with potentially important implications for cancer etiology and response to chemotherapy.

MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites

p53-binding protein 1 (53BP1) is known to be an important mediator of the DNA damage response, with dimethylation of histone H4 lysine 20 (H4K20me2) critical to the recruitment of 53BP1 to double-strand breaks (DSBs). However, it is not clear how 53BP1 is specifically targeted to the sites of DNA damage, as the overall level of H4K20me2 does not seem to increase following DNA damage. It has been proposed that DNA breaks may cause exposure of methylated H4K20 previously buried within the chromosome; however, experimental evidence for such a model is lacking. Here we found that H4K20 methylation actually increases locally upon the induction of DSBs and that methylation of H4K20 at DSBs is mediated by the histone methyltransferase MMSET (also known as NSD2 or WHSC1) in mammals. Downregulation of MMSET significantly decreases H4K20 methylation at DSBs and the subsequent accumulation of 53BP1. Furthermore, we found that the recruitment of MMSET to DSBs requires the γH2AX–MDC1 pathway; specifically, the interaction between the MDC1 BRCT domain and phosphorylated Ser 102 of MMSET. Thus, we propose that a pathway involving γH2AX–MDC1–MMSET regulates the induction of H4K20 methylation on histones around DSBs, which, in turn, facilitates 53BP1 recruitment.

AMPK regulates histone H2B O-GlcNAcylation

Histone H2B O-GlcNAcylation is an important post-translational modification of chromatin during gene transcription. However, how this epigenetic modification is regulated remains unclear. Here we found that the energy-sensing adenosine-monophosphate-activated protein kinase (AMPK) could suppress histone H2B O-GlcNAcylation. AMPK directly phosphorylates O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT). Although this phosphorylation does not regulate the enzymatic activity of OGT, it inhibits OGT–chromatin association, histone O-GlcNAcylation and gene transcription. Conversely, OGT also O-GlcNAcylates AMPK and positively regulates AMPK activity, creating a feedback loop. Taken together, these results reveal a crosstalk between the LKB1-AMPK and the hexosamine biosynthesis (HBP)-OGT pathways, which coordinate together for the sensing of nutrient state and regulation of gene transcription.

A cell cycle-dependent BRCA1–UHRF1 cascade regulates DNA double-strand break repair pathway choice

BRCA1 is an important mediator of the DNA damage response, which promotes homologous recombination (HR) and antagonizes 53BP1-dependent non-homologous end joining in S/G2 phase. But how this is achieved remains unclear. Here, we report that the E3 ubiquitin ligase UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) directly participates in the interplay between BRCA1 and 53BP1. Mechanistically, UHRF1 is recruited to DNA double-strand breaks (DSBs) by BRCA1 in S phase, which requires the BRCT domain of BRCA1 and phosphorylated Ser674 of UHRF1. Subsequently, UHRF1 mediates K63-linked polyubiquitination of RIF1, and results in its dissociation from 53BP1 and DSBs thereby facilitating HR initiation. Thus, UHRF1 is a key regulator of DSB repair choice, which is separate from its role in heterochromatin formation and epigenetic regulator.

The Deubiquitylating Enzyme USP4 Cooperates with CtIP in DNA Double-Strand Break End Resection

DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB) repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR). USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.

Deubiquitination and Activation of AMPK by USP10

The AMP-activated protein kinase (AMPK) is the master regulator of metabolic homeostasis by sensing cellular energy status. When intracellular ATP levels decrease during energy stress, AMPK is initially activated through AMP or ADP binding and phosphorylation of a threonine residue (Thr-172) within the activation loop of its kinase domain. Here we report a key molecular mechanism by which AMPK activation is amplified under energy stress. We found that ubiquitination on AMPKα blocks AMPKα phosphorylation by LKB1. The deubiquitinase USP10 specifically removes ubiquitination on AMPKα to facilitate AMPKα phosphorylation by LKB1. Under energy stress, USP10 activity in turn is enhanced through AMPK-mediated phosphorylation of Ser76 of USP10. Thus, USP10 and AMPK form a key feedforward loop ensuring amplification of AMPK activation in response to fluctuation of cellular energy status. Disruption of this feedforward loop leads to improper AMPK activation and multiple metabolic defects.

Emerging role of FKBP51 in AKT kinase/protein kinase B signaling.

Comment on: Pei H, et al. Cancer Cell 2009; 16:259-66.

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication

ABSTRACT The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4Cdt2) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

And-1 coordinates with CtIP for efficient homologous recombination and DNA damage checkpoint maintenance.

Abstract To prevent genomic instability, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homologous recombination repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of CHK1 kinase to induce the cell cycle checkpoint. But the mechanism is still not fully understood. Here, we establish that And-1, a replisome component, promotes DNA-end resection and DNA repair by homologous recombination. Mechanistically, And-1 interacts with CtIP and regulates CtIP recruitment to DNA damage sites. And-1 localizes to sites of DNA damage dependent on MDC1-RNF8 pathway, and is required for resistance to many DNA-damaging and replication stress-inducing agents. Furthermore, we show that And-1-CtIP axis is critically required for sustained ATR–CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Our findings thus identify And-1 as a novel DNA repair regulator and reveal how the replisome regulates the DNA damage induced checkpoint and genomic stability.