MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor.

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Protective effect of liver X receptor on cigarette smoke and lipopolysaccharide induced airway inflammation and emphysema in mice.

OBJECTIVE: The aim of this study is to assess the impact of Liver X receptors (LXRs) on airway inflammation, airway remodeling, and lipid deposition induced by cigarette smoke and lipopolysaccharide (LPS) exposure in the lung. METHODS: Wild mice and LXR-deficient mice were exposed to cigarette smoke and LPS to induce airway inflammation and remodeling. In addition, some wild mice received intraperitoneal treatment with the LXR agonist GW3965 before exposure to cigarette smoke and LPS. Lung tissue and bronchoalveolar lavage fluid were collected to evaluate airway inflammation, airway remodeling and lipid deposition. RESULTS: Exposure to cigarette smoke and LPS resulted in airway inflammation, emphysema and lipid accumulation in wild mice. These mice also exhibited downregulated LXRα and ABCA1 in the lung. Treatment with GW3965 mitigated inflammation, remodeling and lipid deposition, while the deletion of LXRs exacerbated these effects. Furthermore, GW3965 treatment following exposure to cigarette smoke and LPS increased LXRα and ABCA1 expression and attenuated MyD88 expression in wild mice. CONCLUSION: LXRs demonstrate the potential to mitigate cigarette smoke and LPS- induced airway inflammation, emphysema and lipid disposition in mice.

Identifying autism spectrum disorder using edge-centric functional connectivity.

Brain network analysis is an effective method to seek abnormalities in functional interactions for brain disorders such as autism spectrum disorder (ASD). Traditional studies of brain networks focus on the node-centric functional connectivity (nFC), ignoring interactions of edges to miss much information that facilitates diagnostic decisions. In this study, we present a protocol based on an edge-centric functional connectivity (eFC) approach, which significantly improves classification performance by utilizing the co-fluctuations information between the edges of brain regions compared with nFC to build the classification mode for ASD using the multi-site dataset Autism Brain Imaging Data Exchange I (ABIDE I). Our model results show that even using the traditional machine-learning classifier support vector machine (SVM) on the challenging ABIDE I dataset, relatively high performance is achieved: 96.41% of accuracy, 98.30% of sensitivity, and 94.25% of specificity. These promising results suggest that the eFC can be used to build a reliable machine-learning framework to diagnose mental disorders such as ASD and promote identifications of stable and effective biomarkers. This study provides an essential complementary perspective for understanding the neural mechanisms of ASD and may facilitate future investigations on early diagnosis of neuropsychiatric disorders.

Mapping structural covariance networks of emotional withdrawal symptoms in males with methamphetamine use disorder during abstinence.

Individuals with methamphetamine use disorder (MUD) often experience anxiety and depressive symptoms during abstinence, which can worsen the likelihood of relapse. Thus, it is essential to understand the neuro-mechanism behind methamphetamine use and its associated emotional withdrawal symptoms in order to develop effective clinical strategies. This study aimed to evaluate associations between emotional withdrawal symptoms and structural covariance networks (SCNs) based on cortical thickness (CTh) across the brain. The CTh measures were obtained from Tl-weighted MRI data from a sample of 48 males with MUD during abstinence and 48 male healthy controls. The severity of anxiety and depressive symptoms was assessed by the Hamilton Anxiety Scale (HAMA) and depression (HAMD) scales. Two important nodes belonging to the brain reward system, the right rostral anterior cingulate cortex (rACC) and medial prefrontal cortex (medPFC), were selected as seeds to conduct SCNs and modulation analysis by emotional symptoms. MUDs showed higher structural covariance between the right rACC and regions in the dorsal attention, right frontoparietal, auditory, visual and limbic networks. They also displayed higher structural covariance between the right medPFC and regions in the limbic network. Moreover, the modulation analysis showed that higher scores on HAMA were associated with increased covariance between the right rACC and the left parahippocampal and isthmus cingulate cortex in the default mode network. These outcomes shed light on the complex neurobiological mechanisms underlying methamphetamine use and its associated emotional withdrawal symptoms and may provide new insights into the development of effective treatments for MUD.

Observation and Characterization of Multiple Resonance Modes in a Chiral Helimagnet CrNb3S6.

The chiral helimagnet CrNb3S6 hosts various temperature- and magnetic-field-stabilized chiral soliton lattices (CSLs) and corresponding exotic collective spin resonance modes, which make it an ideal candidate for future magnetic storage/memory and magnon-based information processing. While most studies have focused on characterizing various static spin textures in this chiral helimagnet, its corresponding collective dynamics have rarely been explored. This study systematically investigates the temperature- and magnetic-field-dependent magnetic dynamics of a single crystal of CrNb3S6 using broadband microwave spectroscopy. We observe an optical mode with a temperature-independent mode number in addition to Kittel-like ferromagnetic resonance (FMR) modes in the CSL phase, consistent with the temperature-independent normalized CSL period L(H)/L(0) based on the 1D chiral sine-Gordon model. Furthermore, combining theoretical model fitting and micromagnetic simulation, we provide a detailed phase diagram and temporal-spatial resolution of dynamic modes, which may help to develop high-frequency exchange-coupling-based spintronic devices.

Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators.

Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.

Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling.

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

Stimulating and Manipulating Robust Circularly Polarized Photoluminescence in Achiral Hybrid Perovskites.

Circularly polarized light (CPL) is essential for optoelectronic and chiro-spintronic applications. Hybrid perovskites, as star optoelectronic materials, have demonstrated CPL activity, which is, however, mostly limited to chiral perovskites. Here, we develop a simple, general, and efficient strategy to stimulate CPL activity in achiral perovskites, which possess rich species, efficient luminescence, and tunable bandgaps. With the formation of van der Waals heterojunctions between chiral and achiral perovskites, a nonequilibrium spin population and thus CPL activity are realized in achiral perovskites by receiving spin-polarized electrons from chiral perovskites. The polarization degree of room-temperature CPL in achiral perovskites is at least one order of magnitude higher than in chiral ones. The CPL polarization degree and emission wavelengths of achiral perovskites can be flexibly designed by tuning chemical compositions, operating temperature, or excitation wavelengths. We anticipate that unlimited types of achiral perovskites can be endowed with CPL activity, benefiting their applications in integrated CPL sources and detectors.

[Research on migraine time-series features classification based on small-sample functional magnetic resonance imaging data].

The extraction of neuroimaging features of migraine patients and the design of identification models are of great significance for the auxiliary diagnosis of related diseases. Compared with the commonly used image features, this study directly uses time-series signals to characterize the functional state of the brain in migraine patients and healthy controls, which can effectively utilize the temporal information and reduce the computational effort of classification model training. Firstly, Group Independent Component Analysis and Dictionary Learning were used to segment different brain areas for small-sample groups and then the regional average time-series signals were extracted. Next, the extracted time series were divided equally into multiple subseries to expand the model input sample. Finally, the time series were modeled using a bi-directional long-short term memory network to learn the pre-and-post temporal information within each time series to characterize the periodic brain state changes to improve the diagnostic accuracy of migraine. The results showed that the classification accuracy of migraine patients and healthy controls was 96.94%, the area under the curve was 0.98, and the computation time was relatively shorter. The experiments indicate that the method in this paper has strong applicability, and the combination of time-series feature extraction and bi-directional long-short term memory network model can be better used for the classification and diagnosis of migraine. This work provides a new idea for the lightweight diagnostic model based on small-sample neuroimaging data, and contributes to the exploration of the neural discrimination mechanism of related diseases.

Excessive Iodine Enabled Ultrathin Inorganic Perovskite Growth at the Liquid-Air Interface.

The liquid-air interface offers a platform for the in-plane growth of free-standing materials. However, it is rarely used for inorganic perovskites and ultrathin non-layered perovskites. Herein the liquid-air interfacial synthesis of inorganic perovskite nanosheets (Cs3 Bi2 I9 , Cs3 Sb2 I9 ) is achieved simply by drop-casting the precursor solution with only the addition of iodine. The products are inaccessible without iodine addition. The thickness and lateral size of these nanosheets can be adjusted through the iodine concentration. The high volatility of the iodine spontaneously drives precursors that normally stay in the liquid to the liquid-air interface. The iodine also repairs in situ iodine vacancies during perovskite growth, giving enhanced optical and optoelectronic properties. The liquid-air interfacial growth of ultrathin perovskites provides multi-degree-of-freedom for constructing perovskite-based heterostructures and devices at atomic scale.

Lithographic Multicolor Patterning on Hybrid Perovskites for Nano-Optoelectronic Applications.

Ultrathin hybrid perovskites, with exotic properties and two-dimensional geometry, exhibit great potential in nanoscale optical and optoelectronic devices. However, it is still challenging for them to be compatible with high-resolution patterning technology toward miniaturization and integration applications, as they can be readily damaged by the organic solvents used in standard lithography processes. Here, a flexible three-step method is developed to make high-resolution multicolor patterning on hybrid perovskite, particularly achieved on a single nanosheet. The process includes first synthesis of precursor PbI2 , then e-beam lithography and final conversion to target perovskite. The patterns with linewidth around 150 nm can be achieved, which can be applied in miniature optoelectronic devices and high-resolution displays. As an example, the channel length of perovskite photodetectors can be down to 126 nm. Through deterministic vapor-phase anion exchange, a perovskite nanosheet can not only gradually alter the color of the same pattern in a wide wavelength range, but also display different colors simultaneously. The authors are optimistic that the method can be applied for unlimited perovskite types and device configurations for their high-integrated miniature applications.

AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression.

The high-risk human papillomaviruses (HPV-16, -18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ-containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high-risk E6 but not low-risk E6 expression led to increases in AIB1 transcript levels by real-time RT-PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV-16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.

Protein phosphatase 2A controls ongoing DNA replication by binding to and regulating cell division cycle 45 (CDC45).

Genomic replication is a highly regulated process and represents both a potential benefit and liability to rapidly dividing cells; however, the precise post-translational mechanisms regulating genomic replication are incompletely understood. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that regulates a diverse array of cellular processes. Here, utilizing both a gain-of-function chemical biology approach and loss-of-function genetic approaches to modulate PP2A activity, we found that PP2A regulates DNA replication. We demonstrate that increased PP2A activity can interrupt ongoing DNA replication, resulting in a prolonged S phase. The impaired replication resulted in a collapse of replication forks, inducing dsDNA breaks, homologous recombination, and a PP2A-dependent replication stress response. Additionally, we show that during replication, PP2A exists in complex with cell division cycle 45 (CDC45) and that increased PP2A activity caused dissociation of CDC45 and polymerase α from the replisome. Furthermore, we found that individuals harboring mutations in the PP2A Aα gene have a higher fraction of genomic alterations, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. These results reveal a new function for PP2A in regulating ongoing DNA replication and a potential role for PP2A in the intra-S-phase checkpoint.

NEDD4 expression is associated with breast cancer progression and is predictive of a poor prognosis.

BACKGROUND: A role for neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) in tumorigenesis has been suggested. However, information is lacking on its role in breast tumor biology. The purpose of this study was to determine the role of NEDD4 in the promotion of the growth and progression of breast cancer (BC) and to evaluate the clinicopathologic and prognostic significance of NEDD4. METHODS: The impact of NEDD4 expression in BC cell growth was determined by Cell Counting Kit-8 and colony formation assays. Formalin-fixed paraffin-embedded specimens were collected from 133 adjacent normal tissues (ANTs), 445 BC cases composed of pre-invasive ductal carcinoma in situ (DCIS, n = 37), invasive ductal carcinomas (IDC, n = 408, 226 without and 182 with lymph node metastasis), and 116 invaded lymph nodes. The expression of NEDD4 was analyzed by immunohistochemistry. The association between NEDD4 expression and clinicopathological characteristics was analyzed by chi-square test. Survival was evaluated using the Kaplan-Meier method, and curves were compared using a log-rank test. Univariate and multivariate analyses were performed using the Cox regression method. RESULTS: NEDD4 promoted BC growth in vitro. In clinical retrospective studies, 16.5% of ANTs (22/133) demonstrated positive NEDD4 staining. Strikingly, the proportion of cases showing NEDD4-positive staining increased to 51.4% (19/37) in DCIS, 58.4% (132/226) in IDC without lymph node metastasis, and 73.1% (133/182) in BC with lymph node metastasis (BCLNM). In addition, NEDD4-positive staining was associated with clinical parameters, including tumor size (P = 0.030), nodal status (P = 0.001), estrogen receptor status (P = 0.035), and progesterone receptor status (P = 0.023). Moreover, subset analysis in BCLNM revealed that high NEDD4 expression correlated with an elevated risk of relapse (P = 0.0276). Further, NEDD4 expression was an independent prognostic predictor. Lastly, the rates for 10-year overall survival and disease-free survival were significantly lower in patients with positive NEDD4 staining than those in BC patients with negative NEDD4 staining BC (P = 0.0024 and P = 0.0011, respectively). CONCLUSIONS: NEDD4 expression is elevated in BC and is associated with BC growth. NEDD4 correlated with clinicopathological parameters and predicts a poor prognosis. Thus, NEDD4 is a potential biomarker of poor prognosis and a potential therapeutic target for BC treatment.

Multimodal MRI-based classification of migraine: using deep learning convolutional neural network.

BACKGROUND: Recently, deep learning technologies have rapidly expanded into medical image analysis, including both disease detection and classification. As far as we know, migraine is a disabling and common neurological disorder, typically characterized by unilateral, throbbing and pulsating headaches. Unfortunately, a large number of migraineurs do not receive the accurate diagnosis when using traditional diagnostic criteria based on the guidelines of the International Headache Society. As such, there is substantial interest in developing automated methods to assist in the diagnosis of migraine. METHODS: To the best of our knowledge, no studies have evaluated the potential of deep learning technologies in assisting with the classification of migraine patients. Here, we used deep learning methods in combination with three functional measures (the amplitude of low-frequency fluctuations, regional homogeneity and regional functional correlation strength) based on rs-fMRI data to distinguish not only between migraineurs and healthy controls, but also between the two subtypes of migraine. We employed 21 migraine patients without aura, 15 migraineurs with aura, and 28 healthy controls. RESULTS: Compared with the traditional support vector machine classifier, which has an accuracy of 83.67%, our Inception module-based convolutional neural network approach showed a significant improvement in classification output (over 86.18%). Our data also indicate that the Inception module-based CNN performs better than the AlexNet-based CNN (Inception module-based CNN reached an accuracy of 99.25%). Finally, we also found that regional functional correlation strength (RFCS) could be regarded as the optimum input out of the three indices (ALFF, ReHo, RFCS). CONCLUSIONS: Overall, our study shows that combining the three functional measures of rs-fMRI with deep learning classification is a powerful method to distinguish between migraineurs and healthy individuals. Our data also highlight that deep learning-based frameworks could be used to develop more complicated models or systems to aid in clinical decision making in the future.

Exploration of intrinsic brain activity in migraine with and without comorbid depression.

BACKGROUND: Major depressive disorder is a common comorbidity in migraineurs. Depression may affect the progression and prognosis of migraine. Few studies have examined the brain function in migraineurs that may cause this comorbidity. Here, we aimed to explore depression-related abnormalities in the intrinsic brain activity of interictal migraineurs with comorbid depression using resting-state functional magnetic resonance imaging. RESULTS: Significant main effects of migraine and depression provided evidence that migraine and depression jointly affected the left medial prefrontal cortex, which was thought to be the neural basis of self-referential mental activity in previous studies. Abnormalities in this region may contribute to determining the common symptoms of migraine and depression and even result in comorbidity. Additionally, migraineurs with comorbid depression had different developmental trajectories in the right thalamus and fusiform, which were associated with recognizing, transmitting, controlling and remembering pain and emotion. CONCLUSIONS: Based on our findings, the abnormal mPFC which may contribute to determining the common symptoms in migraine and depression and may be a therapeutic target for migraineurs comorbid depression. The different developmental trajectory in thalamus and fusiform indicates that the comorbidity may arise through a specific mechanism rather than simple superposition of migraine and depression.

[Research of electroencephalography representational emotion recognition based on deep belief networks].

In recent years, with the rapid development of machine learning techniques,the deep learning algorithm has been widely used in one-dimensional physiological signal processing. In this paper we used electroencephalography (EEG) signals based on deep belief network (DBN) model in open source frameworks of deep learning to identify emotional state (positive, negative and neutrals), then the results of DBN were compared with support vector machine (SVM). The EEG signals were collected from the subjects who were under different emotional stimuli, and DBN and SVM were adopted to identify the EEG signals with changes of different characteristics and different frequency bands. We found that the average accuracy of differential entropy (DE) feature by DBN is 89.12%±6.54%, which has a better performance than previous research based on the same data set. At the same time, the classification effects of DBN are better than the results from traditional SVM (the average classification accuracy of 84.2%±9.24%) and its accuracy and stability have a better trend. In three experiments with different time points, single subject can achieve the consistent results of classification by using DBN (the mean standard deviation is1.44%), and the experimental results show that the system has steady performance and good repeatability. According to our research, the characteristic of DE has a better classification result than other characteristics. Furthermore, the Beta band and the Gamma band in the emotional recognition model have higher classification accuracy. To sum up, the performances of classifiers have a promotion by using the deep learning algorithm, which has a reference for establishing a more accurate system of emotional recognition. Meanwhile, we can trace through the results of recognition to find out the brain regions and frequency band that are related to the emotions, which can help us to understand the emotional mechanism better. This study has a high academic value and practical significance, so further investigation still needs to be done.

The Stress-responsive Gene ATF3 Mediates Dichotomous UV Responses by Regulating the Tip60 and p53 Proteins.

The response to UV irradiation is important for a cell to maintain its genetic integrity when challenged by environmental genotoxins. An immediate early response to UV irradiation is the rapid induction of activating transcription factor 3 (ATF3) expression. Although emerging evidence has linked ATF3 to stress pathways regulated by the tumor suppressor p53 and the histone acetyltransferase Tip60, the role of ATF3 in the UV response remains largely unclear. Here, we report that ATF3 mediated dichotomous UV responses. Although UV irradiation enhanced the binding of ATF3 to Tip60, knockdown of ATF3 expression decreased Tip60 stability, thereby impairing Tip60 induction by UV irradiation. In line with the role of Tip60 in mediating UV-induced apoptosis, ATF3 promoted the death of p53-defective cells in response to UV irradiation. However, ATF3 could also activate p53 and promote p53-mediated DNA repair, mainly through altering histone modifications that could facilitate recruitment of DNA repair proteins (such as DDB2) to damaged DNA sites. As a result, ATF3 rather protected the p53 wild-type cells from UV-induced apoptosis. Our results thus indicate that ATF3 regulates cell fates upon UV irradiation in a p53-dependent manner.

H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes.

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.

53BP1 promotes microhomology-mediated end-joining in G1-phase cells.

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.