C1QBP Promotes Homologous Recombination by Stabilizing MRE11 and Controlling the Assembly and Activation of MRE11/RAD50/NBS1 Complex.

MRE11 nuclease forms a trimeric complex (MRN) with RAD50 and NBS1 and plays a central role in preventing genomic instability. When DNA double-strand breaks (DSBs) occur, MRN is quickly recruited to the damage site and initiates DNA end resection; accordingly, MRE11 must be tightly regulated to avoid inefficient repair or nonspecific resection. Here, we show that MRE11 and RAD50 form a complex (MRC) with C1QBP, which stabilizes MRE11/RAD50, while inhibiting MRE11 nuclease activity by preventing its binding to DNA or chromatin. Upon DNA damage, ATM phosphorylates MRE11-S676/S678 to quickly dissociate the MRC complex. Either excess or insufficient C1QBP impedes the recruitment of MRE11 to DSBs and impairs the DNA damage response. C1QBP is highly expressed in breast cancer and positively correlates with MRE11 expression, and the inhibition of C1QBP enhances tumor regression with chemotherapy. By influencing MRE11 at multiple levels, C1QBP is, thus, an important player in the DNA damage response.

PARP1 UFMylation ensures the stability of stalled replication forks.

The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

BRCA1 Promotes Repair of DNA Damage in Cochlear Hair Cells and Prevents Hearing Loss.

Cochlear hair cells (HCs) sense sound waves and allow us to hear. Loss of HCs will cause irreversible sensorineural hearing loss. It is well known that DNA damage repair plays a critical role in protecting cells in many organs. However, how HCs respond to DNA damage and how defective DNA damage repair contributes to hearing loss remain elusive. In this study, we showed that cisplatin induced DNA damage in outer hair cells (OHCs) and promoted OHC loss, leading to hearing loss in mice of either sex. Cisplatin induced the expression of Brca1, a DNA damage repair factor, in OHCs. Deficiency of Brca1 induced OHC and hearing loss, and further promoted cisplatin-induced DNA damage in OHCs, accelerating OHC loss. This study provides the first in vivo evidence demonstrating that cisplatin mainly induces DNA damage in OHCs and that BRCA1 promotes repair of DNA damage in OHCs and prevents hearing loss. Our findings not only demonstrate that DNA damage-inducing agent generates DNA damage in postmitotic HCs but also suggest that DNA repair factors, like BRCA1, protect postmitotic HCs from DNA damage-induced cell death and hearing loss.

PTIP UFMylation promotes replication fork degradation in BRCA1-deficient cells.

Homologous-recombination deficiency due to breast cancer 1/2 (BRCA1/2) mutations or mimicking BRCA1/2 mutations confer synthetic lethality with poly-(ADP)-ribose polymerase 1/2 inhibitors. The chromatin regulator Pax2 transactivation domain interacting protein (PTIP) promotes stalled replication fork degradation in BRCA1-deficient cells, but the underlying mechanism by which PTIP regulates stalled replication fork stability is unclear. Here, we performed a series of in vitro analyses to dissect the function of UFMylation in regulating fork stabilization in BRCA1-deficient cells. By denaturing co-immunoprecipitation, we first found that replication stress can induce PTIP UFMylation. Interestingly, this post-translational modification promotes end resection and degradation of nascent DNA at stalled replication forks in BRCA1-deficient cells. By cell viability assay, we found that PTIP-depleted and UFL1-depleted BRCA1 knockdown cells are less sensitive to poly-(ADP)-ribose polymerase inhibitors than the siRNA targeting negative control BRCA1-deficient cells. These results identify a new mechanism by which PTIP UFMylation confers chemoresistance in BRCA1-deficient cells.

USP11 deubiquitinates E-cadherin and maintains the luminal fate of mammary tumor cells to suppress breast cancer.

Basal-like breast cancer may originate from luminal epithelial or cancerous cells. Inadequately repaired DNA damage impairs luminal differentiation and promotes aberrant luminal to basal trans-differentiation in mammary epithelial cells (MECs). Ubiquitin-specific peptidase 11 (USP11), a deubiquitinase, plays a critical role in DNA damage repair. The role of USP11 in controlling mammary cell differentiation and tumorigenesis remains poorly understood. We generated Usp11 knockout mice and breast cancer cell lines expressing wild-type (WT) and mutant forms of USP11. By using these mutant mice, cell lines, and human USP11-deficient and -proficient breast cancer tissues, we tested how USP11 controls mammary cell fate. We generated Usp11 knock-out mice and found that deletion of Usp11 reduced the expression of E-cadherin and promoted DNA damage in MECs. Overexpression of WT USP11, but not a deubiquitinase-inactive mutant form of USP11, promoted luminal differentiation, enhanced DNA damage repair, and suppressed tumorigenesis in mice. Mechanistically, we found that USP11 enhanced the protein expression of E-cadherin dependent on its deubiquitinase activity and that USP11 deubiquitinated E-cadherin at K738. We discovered that USP11 is bound to E-cadherin through its C-terminal region. In human breast cancers, expression of USP11 was positively correlated with that of E-cadherin, and high USP11 predicted better recurrence-free survival. Our findings provide compelling genetic and biochemical evidence that USP11 not only promotes DNA damage repair but also deubiquitinates E-cadherin and maintains the luminal feature of mammary tumor cells, thereby suppressing luminal breast cancer.

Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

Symmetric control of sister chromatid cohesion establishment.

Besides entrapping sister chromatids, cohesin drives other high-order chromosomal structural dynamics like looping, compartmentalization and condensation. ESCO2 acetylates a subset of cohesin so that cohesion must be established and only be established between nascent sister chromatids. How this process is precisely achieved remains unknown. Here, we report that GSK3 family kinases provide higher hierarchical control through an ESCO2 regulator, CRL4MMS22L. GSK3s phosphorylate Thr105 in MMS22L, resulting in homo-dimerization of CRL4MMS22L and ESCO2 during S phase as evidenced by single-molecule spectroscopy and several biochemical approaches. A single phospho-mimicking mutation on MMS22L (T105D) is sufficient to mediate their dimerization and rescue the cohesion defects caused by GSK3 or MMS22L depletion, whereas non-phosphorylable T105A exerts dominant-negative effects even in wildtype cells. Through cell fractionation and time-course measurements, we show that GSK3s facilitate the timely chromatin association of MMS22L and ESCO2 and subsequently SMC3 acetylation. The necessity of ESCO2 dimerization implicates symmetric control of cohesion establishment in eukaryotes.

The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair.

Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.

ASPM promotes ATR-CHK1 activation and stabilizes stalled replication forks in response to replication stress.

ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.

GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer.

BACKGROUND: Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. RESULTS: Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. CONCLUSIONS: These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

O-GlcNAc has crosstalk with ADP-ribosylation via PARG.

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, a prevalent protein post-translational modification (PTM) that occurs intracellularly, has been shown to crosstalk with phosphorylation and ubiquitination. However, it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that the poly(ADP-ribosyl)ation "eraser", ADP-ribose glycohydrolase (PARG), is O-GlcNAcylated at Ser26, which is in close proximity to its nuclear localization signal. O-GlcNAcylation of PARG promotes nuclear localization and chromatin association. Upon DNA damage, O-GlcNAcylation augments the recruitment of PARG to DNA damage sites and interacting with proliferating cell nuclear antigen (PCNA). In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances the poly(ADP-ribosyl)ation of DNA damage-binding protein 1 (DDB1) and attenuates its auto-ubiquitination, thereby stabilizing DDB1 and allowing it to degrade its downstream targets, such as c-Myc. We further demonstrated that PARG-S26A, the O-GlcNAc-deficient mutant, promoted HCC in mouse xenograft models. Our findings thus reveal that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs.

TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination.

Expression of the E3 ligase TRIM21 is increased in a broad spectrum of cancers; however, the functionally relevant molecular pathway targeted by TRIM21 overexpression remains largely unknown. Here, we show that TRIM21 directly interacts with and ubiquitinates CLASPIN, a mediator for ATR-dependent CHK1 activation. TRIM21-mediated K63-linked ubiquitination of CLASPIN counteracts the K6-linked ubiquitination of CLASPIN which is essential for its interaction with TIPIN and subsequent chromatin loading. We further show that overexpression of TRIM21, but not a TRIM21 catalytically inactive mutant, compromises CHK1 activation, leading to replication fork instability and tumorigenesis. Our findings demonstrate that TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination, providing a potential target for cancer therapy.

Dynamic switching of crotonylation to ubiquitination of H2A at lysine 119 attenuates transcription-replication conflicts caused by replication stress.

The reversible post-translational modification (PTM) of proteins plays an important role in many cellular processes. Lysine crotonylation (Kcr) is a newly identified PTM, but its functional significance remains unclear. Here, we found that Kcr is involved in the replication stress response. We show that crotonylation of histone H2A at lysine 119 (H2AK119) and ubiquitination of H2AK119 are reversibly regulated by replication stress. Decrotonylation of H2AK119 by SIRT1 is a prerequisite for subsequent ubiquitination of H2AK119 by BMI1. Accumulation of ubiquitinated H2AK119 at reversed replication forks leads to the release of RNA Polymerase II and transcription repression in the vicinity of stalled replication forks. These effects attenuate transcription-replication conflicts (TRCs) and TRC-associated R-loop formation and DNA double-strand breaks. These findings suggest that decrotonylation and ubiquitination of H2A at lysine 119 act together to resolve replication stress-induced TRCs and protect genome stability.

TEB/POLQ plays dual roles in protecting Arabidopsis from NO-induced DNA damage.

Nitric oxide (NO) is a key player in numerous physiological processes. Excessive NO induces DNA damage, but how plants respond to this damage remains unclear. We screened and identified an Arabidopsis NO hypersensitive mutant and found it to be allelic to TEBICHI/POLQ, encoding DNA polymerase θ. The teb mutant plants were preferentially sensitive to NO- and its derivative peroxynitrite-induced DNA damage and subsequent double-strand breaks (DSBs). Inactivation of TEB caused the accumulation of spontaneous DSBs largely attributed to endogenous NO and was synergistic to DSB repair pathway mutations with respect to growth. These effects were manifested in the presence of NO-inducing agents and relieved by NO scavengers. NO induced G2/M cell cycle arrest in the teb mutant, indicative of stalled replication forks. Genetic analyses indicate that Polθ is required for translesion DNA synthesis across NO-induced lesions, but not oxidation-induced lesions. Whole-genome sequencing revealed that Polθ bypasses NO-induced base adducts in an error-free manner and generates mutations characteristic of Polθ-mediated end joining. Our experimental data collectively suggests that Polθ plays dual roles in protecting plants from NO-induced DNA damage. Since Polθ is conserved in higher eukaryotes, mammalian Polθ may also be required for balancing NO physiological signaling and genotoxicity.

SETD2-mediated H3K14 trimethylation promotes ATR activation and stalled replication fork restart in response to DNA replication stress.

Ataxia telangiectasia and Rad3 related (ATR) activation after replication stress involves a cascade of reactions, including replication protein A (RPA) complex loading onto single-stranded DNA and ATR activator loading onto chromatin. The contribution of histone modifications to ATR activation, however, is unclear. Here, we report that H3K14 trimethylation responds to replication stress by enhancing ATR activation. First, we confirmed that H3K14 monomethylation, dimethylation, and trimethylation all exist in mammalian cells, and that both SUV39H1 and SETD2 methyltransferases can catalyze H3K14 trimethylation in vivo and in vitro. Interestingly, SETD2-mediated H3K14 trimethylation markedly increases in response to replication stress induced with hydroxyurea, a replication stress inducer. Under these conditions, SETD2-mediated H3K14me3 recruited the RPA complex to chromatin via a direct interaction with RPA70. The increase in H3K14me3 levels was abolished, and RPA loading was attenuated when SETD2 was depleted or H3K14 was mutated. Rather, the cells were sensitive to replication stress such that the replication forks failed to restart, and cell-cycle progression was delayed. These findings help us understand how H3K14 trimethylation links replication stress with ATR activation.

PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair.

Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1-PLK1-RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.

RBM14 promotes DNA end resection during homologous recombination repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.

CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model.

miRNAs are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. C-terminal-binding protein-interacting protein (CtIP) is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous recombination-mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate, and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members that are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed human colon cancer cell line tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer.