FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways.

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection.

The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.

Single-cell analysis of isoform switching and transposable element expression during preimplantation embryonic development.

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.

SlERF.F12 modulates the transition to ripening in tomato fruit by recruiting the co-repressor TOPLESS and histone deacetylases to repress key ripening genes.

Ethylene response factors (ERFs) are downstream components of ethylene-signaling pathways known to play critical roles in ethylene-controlled climacteric fruit ripening, yet little is known about the molecular mechanism underlying their mode of action. Here, we demonstrate that SlERF.F12, a member of the ERF.F subfamily containing Ethylene-responsive element-binding factor-associated Amphiphilic Repression (EAR) motifs, negatively regulates the onset of tomato (Solanum lycopersicum) fruit ripening by recruiting the co-repressor TOPLESS 2 (TPL2) and the histone deacetylases (HDAs) HDA1/HDA3 to repress the transcription of ripening-related genes. The SlERF.F12-mediated transcriptional repression of key ripening-related genes 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2 (ACS2), ACS4, POLYGALACTURONASE 2a, and PECTATE LYASE is dependent on the presence of its C-terminal EAR motif. We show that SlERF.F12 interacts with the co-repressor TPL2 via the C-terminal EAR motif and recruits HDAs SlHDA1 and SlHDA3 to form a tripartite complex in vivo that actively represses transcription of ripening genes by decreasing the level of the permissive histone acetylation marks H3K9Ac and H3K27Ac at their promoter regions. These findings provide new insights into the ripening regulatory network and uncover a direct link between repressor ERFs and histone modifiers in modulating the transition to ripening of climacteric fruit.

Deciphering the Clinical Significance and Kinase Functions of GSK3α in Colon Cancer by Proteomics and Phosphoproteomics.

GSK3α and GSK3ß are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK3ß plays important roles in the pathogenesis of cancer, while GSK3α has long been considered a functionally redundant protein of GSK3ß. Few studies have specifically investigated the functions of GSK3α. In this study, unexpectedly, we found that the expression of GSK3α, but not GSK3ß, was significantly correlated with the overall survival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3α in colon cancer, we profiled the phosphorylation substrates of GSK3α and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3α. A number of these GSK3α-mediated phosphosites have never been reported before or have been incorrectly identified as substrates of GSK3ß. Among them, the levels of HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were significantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3α. The interaction between THRAP3 and GSK3α was verified by biochemical experiments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3α. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.

Integrated screens uncover a cell surface tumor suppressor gene KIRREL involved in Hippo pathway.

Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that Kirrel1 loss markedly promoted tumor growth in vivo. Moreover, KIRREL was significantly enriched in a separate CRISPR screen based on a specific Hippo pathway reporter. Further studies revealed that KIRREL binds directly to SAV1 to activate the Hippo tumor suppressor pathway. Together, our integrated screens reveal a cell surface tumor suppressor involved in the Hippo pathway and highlight the potential of these approaches in biomedical research.

Abro1 maintains genome stability and limits replication stress by protecting replication fork stability.

Protection of the stalled replication fork is crucial for responding to replication stress and minimizing its impact on chromosome instability, thus preventing diseases, including cancer. We found a new component, Abro1, in the protection of stalled replication fork integrity. Abro1 deficiency results in increased chromosome instability, and Abro1-null mice are tumor-prone. We show that Abro1 protects stalled replication fork stability by inhibiting DNA2 nuclease/WRN helicase-mediated degradation of stalled forks. Depletion of RAD51 prevents the DNA2/WRN-dependent degradation of stalled forks in Abro1-deficient cells. This mechanism is distinct from the BRCA2-dependent fork protection pathway, in which stable RAD51 filament formation prevents MRE11-dependent degradation of the newly synthesized DNA at stalled forks. Thus, our data reveal a new aspect of regulated protection of stalled replication forks that involves Abro1.

Wolf-Hirschhorn syndrome candidate 1 (Whsc1) methyltransferase signals via a Pitx2-miR-23/24 axis to effect tooth development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

The MADS-box protein SlTAGL1 regulates a ripening-associated SlDQD/SDH2 involved in flavonoid biosynthesis and resistance against Botrytis cinerea in post-harvest tomato fruit.

3-Dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites is still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers resistance against Botrytis cinerea attack in post-harvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomatoes.

Unbiasedly decoding the tumor microenvironment with single-cell multiomics analysis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor prognosis and limited therapeutic options. Research on the tumor microenvironment (TME) of PDAC has propelled the development of immunotherapeutic and targeted therapeutic strategies with a promising future. The emergence of single-cell sequencing and mass spectrometry technologies, coupled with spatial omics, has collectively revealed the heterogeneity of the TME from a multiomics perspective, outlined the development trajectories of cell lineages, and revealed important functions of previously underrated myeloid cells and tumor stroma cells. Concurrently, these findings necessitated more refined annotations of biological functions at the cell cluster or single-cell level. Precise identification of all cell clusters is urgently needed to determine whether they have been investigated adequately and to identify target cell clusters with antitumor potential, design compatible treatment strategies, and determine treatment resistance. Here, we summarize recent research on the PDAC TME at the single-cell multiomics level, with an unbiased focus on the functions and potential classification bases of every cellular component within the TME, and look forward to the prospects of integrating single-cell multiomics data and retrospectively reusing bulk sequencing data, hoping to provide new insights into the PDAC TME.

Tumour-associated macrophages and Schwann cells promote perineural invasion via paracrine loop in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is frequently accompanied by perineural invasion (PNI), which is associated with excruciating neuropathic pain and malignant progression. However, the relationship between PNI and tumour stromal cells has not been clarified. METHODS: The dorsal root ganglia or sciatic nerves nerve model was used to observe the paracrine interaction and the activation effect among Schwann cells, tumour-associated macrophages (TAMs), and pancreatic cancer cells in vitro. Next generation sequencing, enzyme-linked immunosorbent assay and chromatin immunoprecipitation were used to explore the specific paracrine signalling between TAMs and Schwann cells. RESULTS: We demonstrated that more macrophages were expressed around nerves that have been infiltrated by pancreatic cancer cells compared with normal nerves in murine and human PNI specimens. In addition, high expression of CD68 or GFAP is associated with an increased incidence of PNI and indicates a poor 5-year survival rate in patients with PDAC. Mechanistically, tumour-associated macrophages (TAMs) activate Schwann cells via the bFGF/PI3K/Akt/c-myc/GFAP pathway. Schwann cells secrete IL-33 to recruit macrophages into the perineural milieu and facilitate the M2 pro-tumourigenic polarisation of macrophages. CONCLUSIONS: Our study demonstrates that the bFGF/IL-33 positive feedback loop between Schwann cells and TAMs is essential in the process of PNI of PDAC. The bFGF/PI3K/Akt/c-myc/GFAP pathway would open potential avenues for targeted therapy of PDAC.

ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.

Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.

Crystal structure and function analysis of 6-phosphogluconate dehydrogenase in Mycobacterium tuberculosis.

6-phosphogluconate dehydrogenase (6PGDH) is an essential enzyme in energy metabolism and redox reactions, and represents a potential drug target for the development of therapies targeting trypanosomes, plasmodium, or other pathogens. Tuberculosis, caused by Mycobacterium tuberculosis, is a contagious disease that severely affects human health, with approximately one-third of the world's population infected. However, the protein structure, exact oligomeric state, and catalytic mechanism of 6PGDH in Mycobacterium tuberculosis (Mt6PGDH) have remained largely unknown. In this study, we successfully purified and determined the structure of Mt6PGDH, revealing its function as a tetramer in both solution and crystal states. Through structural comparisons, we clarified the tetramer formation mechanism and the oligomeric organization of short-chain 6PGDHs. Additionally, we identified key residues for coenzyme recognition and catalytic activity. This work not only deepens our understanding of the enzymatic function of Mt6PGDH but also lays a foundation for the development of drugs targeting this enzyme.

Efficacy and safety of immune checkpoint inhibitors in solid tumor patients combined with chronic coronary syndromes or its risk factor: a nationwide multicenter cohort study.

BACKGROUND: Although, immune checkpoint inhibitors (ICIs) have been widely applied in the therapy of malignant tumors, the efficacy and safety of ICIs in patients with tumors and pre-existing CAD, especially chronic coronary syndromes (CCS) or their risk factors (CRF), is not well identified. METHODS: This was a nationwide multicenter observational study that enrolled participants who diagnosed with solid tumors and received ICIs therapy. The main efficacy indicators were progression-free survival (PFS) and overall survival (OS), followed by objective response rate (ORR) and disease control rate (DCR). Safety was assessed by describing treatment-related adverse events (TRAEs) during ICIs therapy evaluated by the Common Terminology Criteria for Adverse Events 5.0 (CTCAE 5.0). RESULTS: In the current research, we retrospectively analyzed the data of 551 patients diagnosed with solid tumors and received ICIs therapy, and these patients were divided into CCS/CRF group and non-CCS/CRF group. Patients with CCS/CRF had more favorable PFS and OS than patients without CCS/CRF (P < 0.001) and the pre-existing CCS/CRF was a protective factor for survival. The ORR (51.8% vs. 39.1%) and DCR (95.8% vs. 89.2%) were higher in CCS/CRF group than in non-CCS/CRF group (P = 0.003, P = 0.006). In this study, there was no significant difference in treatment-related adverse events (TRAEs), including immune-related adverse events (irAEs), between the two groups. CONCLUSIONS: We concluded that ICIs appear to have better efficacy in malignant solid tumor patients with pre-existing CCS/CRF and are not accompanied by more serious irAEs.

Efficacy and safety of tislelizumab plus lenvatinib as first-line treatment in patients with unresectable hepatocellular carcinoma: a multicenter, single-arm, phase 2 trial.

BACKGROUND: Lenvatinib is widely used in treatment of unresectable hepatocellular carcinoma (uHCC), but the benefit of its combination with immunotherapy needs to be verified. This study evaluated the efficacy and safety of tislelizumab plus lenvatinib in systemic treatment-naïve patients with uHCC. METHODS: In this multicenter, single-arm, phase 2 study, systemic treatment-naïve patients with uHCC received tislelizumab 200 mg every three weeks plus lenvatinib (bodyweight ≥ 60 kg: 12 mg; < 60 kg: 8 mg; once daily). Dose-limiting toxicities (DLTs) were evaluated in safety run-in phase to determine whether to enter the expansion phase. The primary endpoint was objective response rate (ORR) assessed by independent review committee (IRC) per Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1). Based on Simon's two-stage design, > 6 responders were needed in stage 1 (n = 30) to continue the study, and ≥ 18 responders were needed by the end of stage 2 (n = 60) to demonstrate statistical superiority to a historical control of lenvatinib monotherapy. RESULTS: Sixty-four patients were enrolled. No DLTs were reported. The study achieved statistical superiority (p = 0.0003) with 23 responders assessed by IRC per RECIST v1.1 in the first 60 patients of the efficacy evaluable analysis set (n = 62). After a median follow-up of 15.7 months, confirmed ORR and disease control rate were 38.7% (24/62, 95% confidence interval [CI], 26.6-51.9) and 90.3% (56/62, 95% CI, 80.1-96.4), respectively. Median progression-free survival was 8.2 months (95% CI, 6.8-not evaluable). Overall survival rate at 12 months was 88.6% (95% CI, 77.7-94.4). Grade ≥ 3 treatment-related adverse events occurred in 18 (28.1%) patients. CONCLUSIONS: Tislelizumab plus lenvatinib demonstrated promising antitumor activity with favourable tolerability as first-line therapy for patients with uHCC. TRIAL REGISTRATION: ClinicalTrials.gov (NCT04401800).

131I Induced In Vivo Proteolysis by Photoswitchable azoPROTAC Reinforces Internal Radiotherapy.

Photopharmacology, incorporating photoswitches such as azobenezes into drugs, is an emerging therapeutic method to realize spatiotemporal control of pharmacological activity by light. However, most photoswitchable molecules are triggered by UV light with limited tissue penetration, which greatly restricts the in vivo application. Here, this study proves that 131I can trigger the trans-cis photoisomerization of a reported azobenezen incorporating PROTACs (azoPROTAC). With the presence of 50 µCi mL-1 131I, the azoPROTAC can effectively down-regulate BRD4 and c-Myc levels in 4T1 cells at a similar level as it does under light irradiation (405 nm, 60 mW cm-2). What's more, the degradation of BRD4 can further benefit the 131I-based radiotherapy. The in vivo experiment proves that intratumoral co-adminstration of 131I (300 µCi) and azoPROTC (25 mg kg-1) via hydrogel not only successfully induce protein degradation in 4T1 tumor bearing-mice but also efficiently inhibit tumor growth with enhanced radiotherapeutic effect and anti-tumor immunological effect. This is the first time that a radioisotope is successfully used as a trigger in photopharmacology in a mouse model. It believes that this study will benefit photopharmacology in deep tissue.

Dynamic radiological features predict pathological response after neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Neoadjuvant immunochemotherapy (NICT) plus esophagectomy has emerged as a promising treatment option for locally advanced esophageal squamous cell carcinoma (LA-ESCC). Pathologic complete response (pCR) is a key indicator associated with great efficacy and overall survival (OS). However, there are insufficient indicators for the reliable assessment of pCR. METHODS: 192 patients with LA-ESCC treated with NICT from December 2019 to October 2023 were recruited. According to pCR status, patients were categorized into pCR group (22.92%) and non-pCR group (77.08%). Radiological features of pretreatment and preoperative CT images were extracted. Logistic and COX regressions were trained to predict pathological response and prognosis, respectively. RESULTS: Four of the selected radiological features were combined to construct an ESCC preoperative imaging score (ECPI-Score). Logistic models revealed independent associations of ECPI-Score and vascular sign with pCR, with AUC of 0.918 in the training set and 0.862 in the validation set, respectively. After grouping by ECPI-Score, a higher proportion of pCR was observed among the high-ECPI group and negative vascular sign. Kaplan Meier analysis demonstrated that recurrence-free survival (RFS) with negative vascular sign was significantly better than those with positive (P = 0.038), but not for OS (P = 0.310). CONCLUSIONS: This study demonstrates dynamic radiological features are independent predictors of pCR for LA-ESCC treated with NICT. It will guide clinicians to make accurate treatment plans.

Dynamic m6A mRNA methylation reveals the involvement of AcALKBH10 in ripening-related quality regulation in kiwifruit.

Kiwifruit ripening is a complex and highly coordinated process that occurs in conjunction with the formation of fruit edible quality. The significance of epigenetic changes, particularly the impact of N6-methyladenosine (m6A) RNA modification on fruit ripening and quality formation, has been largely overlooked. We monitored m6A levels and gene expression changes in kiwifruit at four different stages using LC-MS/MS, MeRIP, RNA-seq, and validated the function of AcALKBH10 through heterologous transgenic expression in tomato. Notable m6A modifications occurred predominantly at the stop codons and the 3' UTRs and exhibited a gradual reduction in m6A levels during the fruit ripening process. Moreover, these m6A modifications in the aforementioned sites demonstrated a discernible inverse relationship with the levels of mRNA abundance throughout the ripening process, suggesting a repression effect of m6A modification in the modulation of kiwifruit ripening. We further demonstrated that AcALKBH10 rather than AcECT9 predominantly regulates m6A levels in ripening-related genes, thereby exerting the regulatory control over the ripening process and the accumulation of soluble sugars and organic acids, ultimately influencing fruit ripening and quality formation. In conclusion, our findings illuminate the epi-regulatory mechanism involving m6A in kiwifruit ripening, offering a fresh perspective for cultivating high-quality kiwifruit with enhanced nutritional attributes.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa.

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites.

BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.