Younghoon Kee

Expanded roles of the Fanconi anemia pathway in preserving genomic stability

Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle.

Cell cycle-dependent chromatin loading of the Fanconi anemia core complex by FANCM/FAAP24

Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. A total of 13 FA proteins are involved in regulating genome surveillance and chromosomal stability. The FA core complex, consisting of 8 FA proteins (A/B/C/E/F/G/L/M), is essential for the monoubiquitination of FANCD2 and FANCI. FANCM is a human ortholog of the archaeal DNA repair protein Hef, and it contains a DEAH helicase and a nuclease domain. Here, we examined the effect of FANCM expression on the integrity and localization of the FA core complex. FANCM was exclusively localized to chromatin fractions and underwent cell cycle-dependent phosphorylation and dephosphorylation. FANCM-depleted HeLa cells had an intact FA core complex but were defective in chromatin localization of the complex. Moreover, depletion of the FANCM binding partner, FAAP24, disrupted the chromatin association of FANCM and destabilized FANCM, leading to defective recruitment of the FA core complex to chromatin. Our results suggest that FANCM is an anchor required for recruitment of the FA core complex to chromatin, and that the FANCM/FAAP24 interaction is essential for this chromatin-loading activity. Dysregulated loading of the FA core complex accounts, at least in part, for the characteristic cellular and developmental abnormalities in FA.

UAF1 is a subunit of multiple deubiquitinating enzyme complexes.

A balance between ubiquitination and deubiquitination regulates numerous cellular processes and pathways, and specific deubiquitinating enzymes often play the decisive role of controlling this balance. We recently reported that the USP1 deubiquitinating enzyme, which regulates the Fanconi anemia pathway by deubiquitinating the central player of the pathway, FANCD2, is activated by the WD40-repeat containing UAF1 protein through formation of a stable USP1/UAF1 protein complex. Here we present the isolation of two novel multisubunit deubiquitinating enzyme complexes containing USP12 and USP46, respectively. Both complexes contain the UAF1 protein as a bona fide subunit. Interestingly, UAF1 regulates the enzymatic activity of both enzyme complexes, suggesting that this activator protein may regulate a subclass of human deubiquitinating enzymes. We postulate that additional WD40-containing proteins may also form complexes with other human deubiquitinating enzymes and thereby regulate their activity and substrate specificity.

The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme

Saccharomyces cerevisiae Rsp5 is an essential HECT ubiquitin ligase involved in several biological processes. To gain further insight into regulation of this enzyme, we identified proteins that copurified with epitope‐tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Overexpression of Ubp2 or Rup1 in the rsp5‐1 mutant elicited a strong growth defect, while overexpression of a catalytically inactive Ubp2 mutant or Rup1 deleted of the UBA domain did not, suggesting an antagonistic relationship between Rsp5 and the Ubp2/Rup1 complex. Consistent with this model, rsp5‐1 temperature sensitivity was suppressed by either ubp2Δ or rup1Δ mutations. Ubp2 reversed Rsp5‐catalyzed substrate ubiquitination in vitro, and Rsp5 and Ubp2 preferentially assembled and disassembled, respectively, K63‐linked polyubiquitin chains. Together, these results indicate that Rsp5 activity is modulated by being physically coupled to the Rup1/Ubp2 deubiquitinating enzyme complex, representing a novel mode of regulation for an HECT ubiquitin ligase.

The Deubiquitinating Enzyme Ubp2 Modulates Rsp5-dependent Lys63-linked Polyubiquitin Conjugates in Saccharomyces cerevisiae

The functions of Lys63-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys63-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. A dramatic increase in Lys63-linked conjugates was observed in ubp2Δ cells. The formation of these was Rsp5-dependent, and ubp2Δ phenotypes could be suppressed by prevention of formation of Lys63 conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys63-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Δ and rup1Δ mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys63 conjugates in ubp2Δ cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys63-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys63-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates.

WDR20 Regulates Activity of the USP12·UAF1 Deubiquitinating Enzyme Complex

The UAF1 (Usp1-associated factor 1) protein binds and stimulates three deubiquitinating enzymes: USP1, USP12, and USP46. Although the USP1·UAF1 complex is required for regulation of the Fanconi anemia (FA) DNA repair pathway, less is known about the USP12·UAF1 and the USP46·UAF1 complexes. To understand further the nature of the USP12 and USP46 complexes, we attempted to identify proteins that interact with the USP12 and USP46 deubiquitinating enzyme complexes. We identified WDR20, a WD40-repeat containing protein, as a common binding partner of UAF1, USP12, and USP46. Further analysis showed that WDR20 associates exclusively with USP12 and USP46, not with USP1. Furthermore, we demonstrate the purification of a ternary USP12·UAF1·WDR20 complex. Interestingly, and consistent with the binding assays, WDR20 stimulated the enzymatic activity of USP12·UAF1, but not of USP1·UAF1. Consistent with our previous report that USP12 and USP46 do not regulate the FA pathway, small interference RNA-mediated depletion of WDR20 protein did not affect the FA pathway or DNA damage responses. We provide a model in which WDR20 serves as a stimulatory subunit for preserving and regulating the activity of the subset of the UAF1·USP complexes.

Regulated degradation of FANCM in the Fanconi anemia pathway during mitosis

The 13 Fanconi anemia (FA) proteins cooperate in a common DNA repair pathway. Eight of these proteins are assembled into a multisubunit E3 ligase called the FA core complex. During S phase, the FA core complex is loaded by the FANCM protein into chromatin where it monoubiquitinates its substrates. In mitosis, the FA core complex is released from FANCM by an unknown mechanism. Here we show that FANCM is hyperphosphorylated and degraded during mitosis. beta-TRCP and Plk1 are the key regulators of FANCM degradation. Nondegradable mutant forms of FANCM retain the FA core complex in the chromatin and disrupt the FA pathway. Our data provide a novel mechanism for the cell cycle-dependent regulation of the FA pathway.

Inhibition of the Nedd8 system sensitizes cells to DNA interstrand cross-linking agents.

The Fanconi anemia pathway is required for repair of DNA interstrand cross-links (ICL). Fanconi anemia pathway–deficient cells are hypersensitive to DNA ICL–inducing drugs such as cisplatin. Conversely, hyperactivation of the Fanconi anemia pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the Fanconi anemia pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL–inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8-conjugating enzymes or treatment with a Nedd8-activating enzyme inhibitor MLN4924, suppressed DNA damage–induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the Fanconi anemia pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells. Mol Cancer Res; 10(3); 369–77. ©2012 AACR.

A Single PXY Motif Located within the Carboxyl Terminus of Spt23p and Mga2p Mediates a Physical and Functional Interaction with Ubiquitin Ligase Rsp5p

Proteasome-dependent processing of the endoplasmic reticulum localized transcription factor Spt23p of Saccharomyces cerevisiae generates its transcriptionally competent form and requires the WW domain containing Rsp5p ubiquitin ligase. Although previous studies documented an Rsp5p-Spt23p association in cells, very little is known about the nature of this interaction. We report here the identification of an imperfect type I WW domain-binding site (LPKY) within the carboxyl-terminal region of Spt23p that is required for Rsp5p binding in vitro and in vivo. Deletion of this motif abrogates Rsp5p-induced ubiquitination of Spt23p in vitro and reduces ubiquitination of the Spt23p precursor in yeast. In addition, the Spt23pΔLPKY mutant is inefficiently processed and is defective at up-regulating target gene (OLE1) expression in cells. Deletion of the corresponding LPKY site within Mga2p, an Spt23p homologue, also abrogates Rsp5p binding and Rsp5p-dependent ubiquitination in vitro as well as Rsp5p binding and Mga2p polyubiquitination in cells. However, the Mga2pΔLPKY mutant undergoes efficient proteasome-dependent processing. These experiments indicate that the LPKY motif of Spt23p is required for Rsp5p binding, Rsp5-induced ubiquitination, proteasome-dependent processing, and its OLE1 inducing function. They also suggest that the LPKY motif of Mga2p is required for Rsp5p binding and ubiquitination, and Rsp5p regulates Mga2p function by a mechanism that is independent of providing the partial degradation signal.

Role of Deubiquitinating Enzymes in DNA Repair.

ABSTRACT Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling.