Ubiquitin E3 Ligase FBXO9 Regulates Pluripotency by Targeting DPPA5 for Ubiquitylation and Degradation.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have unique characteristics where they can both contribute to all three germ layers in vivo and self-renewal indefinitely in vitro. Post-translational modifications of proteins, particularly by the ubiquitin proteasome system (UPS), control cell pluripotency, self-renewal, and differentiation. A significant number of UPS members (mainly ubiquitin ligases) regulate pluripotency and influence ESC differentiation with key elements of the ESC pluripotency network (including the "master" regulators NANOG and OCT4) being controlled by ubiquitination. To further understand the role of the UPS in pluripotency, we performed an RNAi screen during induction of cellular reprogramming and have identified FBXO9 as a novel regulator of pluripotency associated protein DPPA5. Our findings indicate that FBXO9 silencing facilitates the induction of pluripotency through decreased proteasomal degradation of DPPA5. These findings identify FBXO9 as a key regulator of pluripotency.

Small-molecule IKKß activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth.

Activation of inhibitor of nuclear factor NF-κB kinase subunit-ß (IKKß), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKß knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKß and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKß is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKß in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKß inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKß but inhibited the IKKß kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKß phosphorylation was inhibited by 39-100, thus we termed it IKKß activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKß levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKß activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

USP11 mediates repair of DNA-protein cross-links by deubiquitinating SPRTN metalloprotease.

DNA-protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.

The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

SOSS complexes participate in the maintenance of genomic stability.

Proteins that bind to single-stranded DNA (ssDNA) are essential for DNA replication, recombinational repair, and maintenance of genomic stability. Here, we describe the characterization of an ssDNA-binding heterotrimeric complex, SOSS (sensor of ssDNA) in human, which consists of human SSB homologs hSSB1/2 (SOSS-B1/2) and INTS3 (SOSS-A) and a previously uncharacterized protein C9orf80 (SOSS-C). We have shown that SOSS-A serves as a central adaptor required not only for SOSS complex assembly and stability, but also for facilitating the accumulation of SOSS complex to DNA ends. Moreover, SOSS-depleted cells display increased ionizing radiation sensitivity, defective G2/M checkpoint, and impaired homologous recombination repair. Thus, our study defines a pathway involving the sensing of ssDNA by SOSS complex and suggests that this SOSS complex is likely involved in the maintenance of genome stability.

The annealing helicase HARP protects stalled replication forks.

Mutations in HepA-related protein (HARP) are the only identified causes of Schimke immunoosseous dysplasia (SIOD). HARP has a unique annealing helicase activity in vitro, but the in vivo functional significance remains unknown. Here, we demonstrated that HARP is recruited to stalled replication forks via its direct interaction with Replication protein A (RPA). Cells with HARP depletion displayed increased spontaneous DNA damage and G2/M arrest, suggesting that HARP normally acts to stabilize stalled replication forks. Our data place the annealing helicase activity of HARP at replication forks and propose that SIOD syndrome may be caused by the destabilization of replication forks during cell proliferation.

Alpha thalassemia/mental retardation syndrome X-linked gene product ATRX is required for proper replication restart and cellular resistance to replication stress.

Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a member of the SWI/SNF protein family of DNA-dependent ATPases. It functions as a chromatin remodeler and is classified as an SNF2-like helicase. Here, we showed somatic knock-out of ATRX displayed perturbed S-phase progression as well as hypersensitivity to replication stress. ATRX is recruited to sites of DNA damage, required for efficient checkpoint activation and faithful replication restart. In addition, we identified ATRX as a binding partner of MRE11-RAD50-NBS1 (MRN) complex. Together, these results suggest a non-canonical function of ATRX in guarding genomic stability.

Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction.

Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero-dimerization. We found that TLK1 and 2 hyper-autophosphorylation suppresses their recruitment to damaged chromatin. Furthermore, both TLK1 and 2 associate with PCNA specifically through their evolutionarily conserved non-canonical PCNA-interacting protein (PIP) box at the N-terminus, and mutation of the PIP-box abolishes their recruitment to DNA damage sites. Mechanistically, the TLK1 and 2 hyper-autophosphorylation masks the PIP-box and negatively regulates their recruitment to the DNA damage site. Overall, our study dissects the detailed genetic regulation of TLK1 and 2 at damaged chromatin, which provides important insights into their emerging roles in DNA repair.

Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis.

DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis (TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase η (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion.

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay.

DNA-protein crosslinks (DPCs) are steric hindrances to DNA metabolic processes and the removal and repair of DPCs is a rapidly evolving area of research. A critical component of deciphering this repair pathway is developing techniques that detect and quantify specific types of DPCs in cells. Here we describe a protocol for direct detection of enzymatic DPCs from mammalian cells-the RADAR assay. The method involves isolating genomic DNA and DPCs from cells and binding them to nitrocellulose membrane with a vacuum slot blot manifold. DPCs are detected using antibodies raised against the protein of interest and quantified by normalizing to a DNA loading control. The RADAR assay allows for the detection of specific types of DPCs and the sensitive analysis of the DNA-protein crosslinking activity of various drugs, is adaptable across different cell types and conditions, and requires little specialized equipment.

Modeling Ewing Sarcoma Lung Metastasis.

Ewing Sarcoma (EwS) is the second most common malignant bone tumor in adolescents and young adults. The single-most powerful predictor of outcome in EwS is presence of metastatic burden at the time of diagnosis. Patients with metastatic Ewing Sarcoma have an abysmal 5-year survival rate of 10%-25%, which has not changed over the past 30-40 years. Thus, unraveling underlying mechanisms of EwS metastasis are imperative for developing effective therapeutic measures. Investigations towards this goal are limited by the lack of reliable genetically engineered mouse models and specialized metastatic models. Using two established cell lines, A673 and TC71, we generated lung specific metastatic cell lines by serial orthotopic intra-tibial injection followed by isolation of cells from lung metastases. The lung metastatic lines generated exhibit distinct differential molecular signatures from the parental cells when analyzed using a multi-omics approach. These signatures overlapped with EwS patient primary bone and metastatic lung specimens supporting the clinical relevance of these preclinical models of EwS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Intra-Tibial injection in NSG mice Basic Protocol 2: Development and characterization of lung metastatic cell line.

FBXO21 mediated degradation of p85α regulates proliferation and survival of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid blasts in the bone marrow (BM). Despite advances in therapy, the prognosis for AML patients remains poor, and there is a need to identify novel molecular pathways regulating tumor cell survival and proliferation. F-box ubiquitin E3 ligase, FBXO21, has low expression in AML, but expression correlates with survival in AML patients and patients with higher expression have poorer outcomes. Silencing FBXO21 in human-derived AML cell lines and primary patient samples leads to differentiation, inhibition of tumor progression, and sensitization to chemotherapy agents. Additionally, knockdown of FBXO21 leads to up-regulation of cytokine signaling pathways. Through a mass spectrometry-based proteomic analysis of FBXO21 in AML, we identified that FBXO21 ubiquitylates p85α, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) pathway, for degradation resulting in decreased PI3K signaling, dimerization of free p85α and ERK activation. These findings reveal the ubiquitin E3 ligase, FBXO21, plays a critical role in regulating AML pathogenesis, specifically through alterations in PI3K via regulation of p85α protein stability.

EHD1-dependent traffic of IGF-1 receptor to the cell surface is essential for Ewing sarcoma tumorigenesis and metastasis.

Overexpression of EPS15 Homology Domain containing 1 (EHD1) has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD mRNA expression specifying shorter patient survival. ShRNA and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified the IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

EHD1-dependent traffic of IGF-1 receptor to the cell surface is essential for Ewing sarcoma tumorigenesis and metastasis.

Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

USP1 Expression Driven by EWS::FLI1 Transcription Factor Stabilizes Survivin and Mitigates Replication Stress in Ewing Sarcoma.

In this study, we identify USP1 as a transcriptional target of EWS::FLI1 and demonstrate the requisite function of USP1 in Ewing sarcoma (EWS) cell survival in response to endogenous replication stress. EWS::FLI1 oncogenic transcription factor drives most EWS, a pediatric bone cancer. EWS cells display elevated levels of R-loops and replication stress. The mechanism by which EWS cells override activation of apoptosis or cellular senescence in response to increased replication stress is not known. We show that USP1 is overexpressed in EWS and EWS::FLI1 regulates USP1 transcript levels. USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. Mechanistically, USP1 regulates Survivin (BIRC5/API4) protein stability and the activation of caspase-9 and caspase-3/7 in response to endogenous replication stress. Notably, USP1 inhibition sensitizes cells to doxorubicin and etoposide treatment. Together, our study demonstrates that USP1 is regulated by EWS::FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes cells to standard of care chemotherapy. IMPLICATIONS: High USP1 and replication stress levels driven by EWS::FLI1 transcription factor in EWS are vulnerabilities that can be exploited to improve existing treatment avenues and overcome drug resistance.

Mechanisms and Regulation of DNA-Protein Crosslink Repair During DNA Replication by SPRTN Protease.

DNA-protein crosslinks (DPCs) are deleterious DNA lesions that occur when proteins are covalently crosslinked to the DNA by the action of variety of agents like reactive oxygen species, aldehydes and metabolites, radiation, and chemotherapeutic drugs. Unrepaired DPCs are blockades to all DNA metabolic processes. Specifically, during DNA replication, replication forks stall at DPCs and are vulnerable to fork collapse, causing DNA breakage leading to genome instability and cancer. Replication-coupled DPC repair involves DPC degradation by proteases such as SPRTN or the proteasome and the subsequent removal of DNA-peptide adducts by nucleases and canonical DNA repair pathways. SPRTN is a DNA-dependent metalloprotease that cleaves DPC substrates in a sequence-independent manner and is also required for translesion DNA synthesis following DPC degradation. Biallelic mutations in SPRTN cause Ruijs-Aalfs (RJALS) syndrome, characterized by hepatocellular carcinoma and segmental progeria, indicating the critical role for SPRTN and DPC repair pathway in genome maintenance. In this review, we will discuss the mechanism of replication-coupled DPC repair, regulation of SPRTN function and its implications in human disease and cancer.

New answers to the old RIDDLE: RNF168 and the DNA damage response pathway.

The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination plays an integral role in regulating cellular processes including DNA damage signaling and repair. The ubiquitin E3 ligase RNF168 is essential in assembling a cohort of DNA repair proteins at the damaged chromatin via its enzymatic activity. RNF168 ubiquitinates histone H2A(X) at the N terminus and generates a specific docking scaffold for ubiquitin-binding motif-containing proteins. The regulation of RNF168 at damaged chromatin and the mechanistic implication in the recruitment of DNA repair proteins to the damaged sites remain an area of active investigation. Here, we review the function and regulation of RNF168 in the context of ubiquitin-mediated DNA damage signaling and repair. We will also discuss the unanswered questions that require further investigation and how understanding RNF168 targeting specificity could benefit the therapeutic development for cancer treatment.

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity.

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.

Selective binding of meiosis-specific yeast Hop1 protein to the holliday junctions distorts the DNA structure and its implications for junction migration and resolution.

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex (SC), plays an important role in both gene conversion and crossing over between homologs, as well as enforces meiotic recombination checkpoint control over the progression of recombination intermediates. In hop1Delta mutants, meiosis-specific double-strand breaks (DSBs) are reduced to 10% of the wild-type level, and at aberrantly late times, these DSBs are processed into inter-sister recombination intermediates. However, the underlying mechanism by which Hop1 protein regulates these nuclear events remains obscure. Here we show that Hop1 protein interacts selectively with the Holliday junction, changes its global conformation and blocks the dissolution of the junction by a RecQ helicase. The Holliday junction-Hop1 protein complexes are significantly more stable at higher ionic strengths and molar excess of unlabeled competitor DNA than complexes containing other recombination intermediates. Structural analysis of the Holliday junction using 2-aminopurine fluorescence emission, DNase I footprinting and KMnO4 probing provide compelling evidence that Hop1 protein binding induces significant distortion at the center of the Holliday junction. We propose that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates with the process of branch migration of Holliday junction.