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Background: Exploring potential biomarkers for predicting clinical outcomes and developing targeted therapies for acute myeloid leukemia (AML) is of utmost importance. This study aimed to investigate the expression pattern of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) pathway and its role in the prognosis of AML patients. Methods: In this study, we examined the prognostic value of TXNIP/NLRP3 pathway in AML patients using microarray data from Gene Expression Omnibus (GEO) and transcriptome data from the Cancer Genome Atlas (TCGA) to develop a prognostic model and validated the results by quantitative real-time PCR (qRT-PCR) in a validation cohort of 26 AML patients and 18 healthy individuals from Jinan University (JNU) database. Results: Analysis of the GSE13159 database revealed that TXNIP , interleukin 1 beta ( IL1B ) within the TXNIP/NLRP3 pathway were significantly upregulated and caspase1 ( CASP1 ) was downregulated in AML patients ( TXNIP , P = 0.031; IL1B , P = 0.042; CASP1 , P = 0.038). Compared to high NLRP3 expression, AML patients with low NLRP3 expression had a longer overall survival (OS) in the GSE12417 dataset ( P = 0.004). Moreover, both the training and validation results indicated that lower TXNIP , NLRP3 , and IL1B expression were associated with favorable prognosis (GSE12417, P = 0.009; TCGA, P = 0.050; JNU, P = 0.026). According to the receiver operating characteristic curve analysis, this model demonstrated a sensitivity of 84% for predicting three-year survival. These data might provide novel predictors for AML outcome and direction for further investigation of the possibility of using TXNIP / NLRP3 / IL1B genes in novel targeted therapies for AML., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Folate receptor (FR) overexpression in a wide range of solid tumors provides an opportunity to develop novel, targeted cancer therapeutics. In this study, we investigated whether prebinding the chemotherapeutic methotrexate (MTX) to folate-binding protein (FBP), the soluble form of FR, would enable the protein to serve as a targeted therapeutic vector, enhancing uptake into tumor cells and improving therapeutic efficacy. In an in vivo study, using an FR-overexpressing KB xenograft model in SCID mice, modest improvement in inhibiting tumor growth was observed for the MTX/FBP mixtures as compared to saline control and free MTX. Surprisingly, FBP alone inhibited tumor growth compared to saline control, free MTX, and FBP/MTX. In order to better understand this effect, we investigated the cytotoxicity of micromolar concentrations of FBP in vitro using the KB, HeLa, and A549 cancer cell lines. Our results revealed concentration-dependent apoptosis (24 h; 10-50 μM) in all three cell lines accompanied by a time- and concentration-dependent reduction (6, 12, and 24 h; 10-50 μM) in metabolic activity and compromised cell plasma membrane integrity. This study demonstrates an apoptosis pathway for cytotoxicity of FBP, an endogenous serum protein, in cancer cell lines with widely varying levels of FR expression. Furthermore, in vivo tumor growth suppression for xenograft KB tumors in SCID mice was observed. These studies suggest novel strategies for the elimination of cancer cells employing endogenous, serum transport proteins.

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF-ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF-ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF-ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4-XPF-ERCC1 complex in ICL repair., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. SPIN1 harbors three Tudor domains, two of which engage the tail of histone H3 by reading the H3-Lys-4 trimethylation and H3-Arg-8 asymmetric dimethylation marks. To gain mechanistic insight into how SPIN1 functions as a transcriptional coactivator, here we purified its interacting proteins. We identified an uncharacterized protein (C11orf84), which we renamed SPIN1 docking protein (SPIN·DOC), that directly binds SPIN1 and strongly disrupts its histone methylation reading ability, causing it to disassociate from chromatin. The Spindlin family of coactivators has five related members (SPIN1, 2A, 2B, 3, and 4), and we found that all of them bind SPIN·DOC. It has been reported previously that SPIN1 regulates gene expression in the Wnt signaling pathway by directly interacting with transcription factor 4 (TCF4). We observed here that SPIN·DOC associates with TCF4 in a SPIN1-dependent manner and dampens SPIN1 coactivator activity in TOPflash reporter assays. Furthermore, knockdown and overexpression experiments indicated that SPIN·DOC represses the expression of a number of SPIN1-regulated genes, including those encoding ribosomal RNA and the cytokine IL1B. In conclusion, we have identified SPIN·DOC as a transcriptional repressor that binds SPIN1 and masks its ability to engage the H3-Lys-4 trimethylation activation mark., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined c lustered, r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cancer stem cells (CSCs) contribute to tumour heterogeneity, therapy resistance and metastasis. However, the regulatory mechanisms of cancer cell stemness remain elusive. Here we identify PCNA-associated factor (PAF) as a key molecule that controls cancer cell stemness. PAF is highly expressed in breast cancer cells but not in mammary epithelial cells (MECs). In MECs, ectopic expression of PAF induces anchorage-independent cell growth and breast CSC marker expression. In mouse models, conditional PAF expression induces mammary ductal hyperplasia. Moreover, PAF expression endows MECs with a self-renewing capacity and cell heterogeneity generation via Wnt signalling. Conversely, ablation of endogenous PAF induces the loss of breast cancer cell stemness. Further cancer drug repurposing approaches reveal that NVP-AUY922 downregulates PAF and decreases breast cancer cell stemness. Our results unveil an unsuspected role of the PAF-Wnt signalling axis in modulating cell plasticity, which is required for the maintenance of breast cancer cell stemness.

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway., (© 2014 Wang et al.; Published by Cold Spring Harbor Laboratory Press.)

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847., (© 2014 Barton et al.)

Human TopBP1 is a key mediator protein involved in DNA replication checkpoint control. In this study, we report a specific interaction between TopBP1 and Bloom syndrome helicase (BLM) that is phosphorylation and cell-cycle dependent. Interestingly, TopBP1 depletion led to decreased BLM protein level and increased sister chromatid exchange (SCE). Moreover, our data indicated that BLM was ubiquitinated by E3 ligase MIB1 and degraded in G1 cells but was stabilized by TopBP1 in S phase cells. Depletion of MIB1 restored BLM protein level and rescued the elevated SCE phenotype in TopBP1-depleted cells. In addition, cells expressing an undegradable BLM mutant showed radiation sensitivity, probably by triggering end resection and inhibiting the nonhomologous end-joining (NHEJ) pathway in G1 phase. Altogether, these data suggest that, although BLM is downregulated in G1 phase in order to promote NHEJ-mediated DNA repair, it is stabilized by TopBP1 in S phase cells in order to suppress SCE and thereby prevent genomic instability., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Activation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The RAD51 recombinase plays a central role in homologous recombination (HR), which is critical for repair of DNA double-strand breaks, maintenance of genomic stability, and prevention of developmental disorders and cancer. Here, we report the identification of an RAD51-binding protein fidgetin-like 1 (FIGNL1). FIGNL1 specifically interacts with RAD51 through its conserved RAD51 binding domain. Cells depleted of FIGNL1 show defective HR repair. Interestingly, FIGNL1 is recruited to sites of DNA damage in a manner that is independent of breast cancer 2, early onset, RAD51, and probably, RAD51 paralogs. Conversely, FIGNL1 depletion does not affect the loading of RAD51 onto ssDNA. Our additional analysis uncovered KIAA0146, also known as scaffolding protein involved in DNA repair (SPIDR), as a binding partner of FIGNL1 and established that KIAA0146/SPIDR acts with FIGNL1 in HR repair. Collectively, our study uncovers a protein complex, which consists of FIGNL1 and KIAA0146/SPIDR, in DNA repair and provides potential directions for cancer diagnosis and therapy.

Mutation of PHF6, which results in the X-linked mental retardation disorder Börjeson-Forssman-Lehmann syndrome, is also present in about 38% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. However, it remains to be determined exactly how PHF6 acts in vivo and what functions of PHF6 may be associated with its putative tumor suppressor function. Here, we demonstrate that PHF6 is a nucleolus, ribosomal RNA promoter-associated protein. PHF6 directly interacts with upstream binding factor (UBF) through its PHD1 domain and suppresses ribosomal RNA (rRNA) transcription by affecting the protein level of UBF. Knockdown of PHF6 impairs cell proliferation and arrests cells at G(2)/M phase, which is accompanied by an increased level of phosphorylated H2AX, indicating that PHF6 deficiency leads to the accumulation of DNA damage in the cell. We found that increased DNA damage occurs at the ribosomal DNA (rDNA) locus in PHF6-deficient cells. This effect could be reversed by knocking down UBF or overexpressing RNASE1, which removes RNA-DNA hybrids, suggesting that there is a functional link between rRNA synthesis and genomic stability at the rDNA locus. Together, these results reveal that the key function of PHF6 is involved in regulating rRNA synthesis, which may contribute to its roles in cell cycle control, genomic maintenance, and tumor suppression.

Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UV(s)S syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UV(s)S syndrome.

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage signaling pathways. The BRCT domain-containing protein BRCTx has been shown to interact physically with RAD18, an E3 ligase involved in postreplication repair and homologous recombination repair. However, the physiological relevance of the interaction between RAD18 and BRCTx is largely unknown. In this study, we showed that RAD18 interacts with BRCTx in a phosphorylation-dependent manner and that this interaction, mediated via highly conserved serine residues on the RAD18 C terminus, is required for BRCTx accumulation at DNA damage sites. Furthermore, we uncovered critical roles of the RAD18-BRCTx module in UV-induced DNA damage repair but not PCNA mono-ubiquitination or homologous recombination. Thus, our results suggest that RAD18 has an additional function in the surveillance of the UV-induced DNA damage response signal., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1-MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.

The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.

In both Drosophila and mammalian systems, the Hippo pathway plays an important role in controlling organ size, mainly through its ability to regulate cell proliferation and apoptosis. The key component in the Hippo pathway is the Yes-associated protein YAP1, which localizes in nucleus, functions as a transcriptional coactivator, and regulates the expression of several proliferation- and apoptosis-related genes. The Hippo pathway negatively regulates YAP1 transcriptional activity by modulating its nuclear-cytoplasmic localization in a phosphorylation-dependent manner. Here, we describe the identification of several new PY motif-containing proteins, including angiomotin-like protein 1 (AMOTL1) and 2 (AMOTL2), as YAP1-associated proteins. We demonstrate that AMOTL1 and AMOTL2 can regulate YAP1 cytoplasm-to-nucleus translocation through direct protein-protein interaction, which can occur independent of YAP1 phosphorylation status. Moreover, down-regulation of AMOTL2 in MCF10A cells promotes epithelial-mesenchymal transition, a phenotype that is also observed in MCF10A cells with YAP1 overexpression. Together, these data support a new mechanism for YAP1 regulation, which is mediated via its direct interactions with angiomotin-like proteins.

Human TopBP1 plays a critical role in the control of DNA replication checkpoint. In this study, we report a specific interaction between TopBP1 and BACH1/FANCJ, a DNA helicase involved in the repair of DNA crosslinks. The TopBP1/BACH1 interaction is mediated by the very C-terminal tandem BRCT domains of TopBP1 and S phase-specific phosphorylation of BACH1 at Thr 1133 site. Interestingly, we demonstrate that depletion of TopBP1 or BACH1 attenuates the loading of RPA on chromatin. Moreover, both TopBP1 and BACH1 are required for ATR-dependent phosphorylation events in response to replication stress. Taken together, our data suggest that BACH1 has an unexpected early role in replication checkpoint control. A specific interaction between TopBP1 and BACH1 is likely to be required for the extension of single-stranded DNA regions and RPA loading following replication stress, which is a prerequisite for the subsequent activation of replication checkpoint.

DNA double-strand breaks (DSBs) represent one of the most lethal types of DNA damage cells encounter. CtIP (also known as RBBP8) acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end resection and the generation of single-stranded DNA, which is critically important for homologous recombination repair. However, it is not yet clear exactly how CtIP participates in this process. Here, we demonstrate that besides the known conserved C terminus, the N terminus of CtIP protein is also required in DSB end resection and DNA damage-induced G(2)/M checkpoint control. We further show that both termini of CtIP can interact with the MRN complex and that the N terminus of CtIP, especially residues 22-45, binds to MRN and plays a critical role in targeting CtIP to sites of DNA breaks. Collectively, our results highlight the importance of the N terminus of CtIP in directing its localization and function in DSB repair.

The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization.

Genomic stability in eukaryotic cells is maintained by the coordination of multiple cellular events including cell cycle checkpoint, DNA repair, transcription, and apoptosis after DNA damage. Pax2 transactivation domain interaction protein (PTIP), a protein that contains six BRCT domains, has been implicated in DNA damage response. In this study we showed that recruitment of PTIP to damaged chromatin depends on DNA damage signaling proteins gammaH2AX.MDC1.RNF8, which in turn facilitates sustained localization of PA1 (PTIP-associated protein 1) to sites of DNA break. Similar to PTIP, depletion of PA1 increases cellular sensitivity to ionizing radiation. Furthermore, we demonstrated that the N-terminal PA1 binding domain and the C-terminal focus-localization domain of PTIP are critical for PTIP function in DNA damage repair. Interestingly, although PTIP and PA1 associate with MLL (mixed lineage leukemia) complexes and participate in transcriptional regulation, this function of PTIP.PA1 in DNA damage response is likely to be independent of the MLL complexes. Taken together, we propose that a subset of PTIP.PA1 complex is recruited to DNA damage sites via the RNF8-dependent pathway and is required for cell survival in response to DNA damage.

The product of the breast cancer-1 gene, BRCA1, plays a crucial part in the DNA damage response through its interactions with many proteins, including BACH1, CtIP and RAP80. Here we identify a coiled-coil domain-containing protein, CCDC98, as a BRCA1-interacting protein. CCDC98 colocalizes with BRCA1 and is required for the formation of BRCA1 foci in response to ionizing radiation. Moreover, like BRCA1, CCDC98 has a role in radiation sensitivity and damage-induced G2/M checkpoint control. Together, these results suggest that CCDC98 is a mediator of BRCA1 function involved in the mammalian DNA damage response.

Mutations in the breast cancer susceptibility gene 1 (BRCA1) are associated with an increased risk of breast and ovarian cancers. BRCA1 participates in the cellular DNA damage response. We report the identification of receptor-associated protein 80 (RAP80) as a BRCA1-interacting protein in humans. RAP80 contains a tandem ubiquitin-interacting motif domain, which is required for its binding with ubiquitin in vitro and its damage-induced foci formation in vivo. Moreover, RAP80 specifically recruits BRCA1 to DNA damage sites and functions with BRCA1 in G2/M checkpoint control. Together, these results suggest the existence of a ubiquitination-dependent signaling pathway involved in the DNA damage response.

Histone lysine methylation has been linked to the recruitment of mammalian DNA repair factor 53BP1 and putative fission yeast homolog Crb2 to DNA double-strand breaks (DSBs), but how histone recognition is achieved has not been established. Here we demonstrate that this link occurs through direct binding of 53BP1 and Crb2 to histone H4. Using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we show that, despite low amino acid sequence conservation, both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2). The structure of 53BP1/H4-K20me2 complex uncovers a unique five-residue 53BP1 binding cage, remarkably conserved in the structure of Crb2, that best accommodates a dimethyllysine but excludes a trimethyllysine, thus explaining the methylation state-specific recognition of H4-K20. This study reveals an evolutionarily conserved molecular mechanism of targeting DNA repair proteins to DSBs by direct recognition of H4-K20me2.

BRCA1 (Breast Cancer Susceptibility Gene 1) possesses an N-terminal Ring domain and tandem C-terminal BRCT motifs. While the Ring domain has E3 ubiquitin ligase activity, the BRCA1 BRCT domains specifically recognize phospho-serine motifs. Here, we demonstrate that BRCA1 Ring domain catalyzes CtIP ubiquitination in a manner that depends on a phosphorylation-mediated interaction between CtIP and BRCA1 BRCT domains. The BRCA1-dependent ubiquitination of CtIP does not target CtIP for degradation. Instead, ubiquitinated CtIP associates with chromatin following DNA damage and participates in G2/M checkpoint control. Thus, we propose that BRCA1 can regulate the functions of its substrates through nonproteasomal pathways that do not involve substrate degradation.

Cell cycle checkpoints are essential for maintaining genomic integrity. Human topoisomerase II binding protein 1 (TopBP1) shares sequence similarity with budding yeast Dpb11, fission yeast Rad4/Cut5, and Xenopus Cut5, all of which are required for DNA replication and cell cycle checkpoints. Indeed, we have shown that human TopBP1 participates in the activation of replication checkpoint and DNA damage checkpoints, following hydroxyurea treatment and ionizing radiation. In this study, we address the physiological function of TopBP1 in S phase by using small interfering RNA. In the absence of exogenous DNA damage, TopBP1 is recruited to replicating chromatin. However, TopBP1 does not appear to be essential for DNA replication. TopBP1-deficient cells have increased H2AX phosphorylation and ATM-Chk 2 activation, suggesting the accumulation of DNA double-strand breaks in the absence of TopBP1. This leads to formation of gaps and breaks at fragile sites, 4N accumulation, and aberrant cell division. We propose that the cellular function of TopBP1 is to monitor ongoing DNA replication. By ensuring proper DNA replication, TopBP1 plays a critical role in the maintenance of genomic stability during normal S phase as well as following genotoxic stress.

The BRCA1 C-terminal (BRCT) domain has recently been implicated as a phospho-protein binding domain. We demonstrate here that a CTBP-interacting protein CtIP interacts with BRCA1 BRCT domains in a phosphorylation-dependent manner. The CtIP/BRCA1 complex only exists in G(2) phase and is required for DNA damage-induced Chk1 phosphorylation and the G(2)/M transition checkpoint. However, the CtIP/BRCA1 complex is not required for the damage-induced G(2) accumulation checkpoint, which is controlled by a separate BRCA1/BACH1 complex. Taken together, these data not only implicate CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage.

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.

Claspin is a newly identified protein that regulates Chk1 activation in Xenopus. In the present study we investigated the role of human Claspin in the DNA damage/replication checkpoint in mammalian cells. We observed that human Claspin is a cell cycle regulated protein that peaks at S/G2 phase. Claspin localizes in the nuclei, but it only associates with Chk1 following replication stress or other types of DNA damage. In addition, Claspin is phosphorylated in response to replication stress, and this phosphorylation appears to be required for its association with Chk1. Moreover, Claspin interacts with the checkpoint proteins ATR and Rad9. Given that both the ATR and Rad9-Rad1-Hus1 complexes are involved in Chk1 activation, it is possible that Claspin works as an adaptor molecule bringing these molecules together. Using small interfering RNA technology, we have shown that down-regulation of Claspin expression inhibits Chk1 activation in response to replication stress. More importantly, down-regulation of Claspin augments the premature chromatin condensation induced by hydroxyurea, inhibits the UV-induced reduction of DNA synthesis, and decreases cell survival. Taken together, these data imply a potentially critical role for Claspin in replication checkpoint control in mammalian cells.

Cell cycle checkpoints play a central role in genomic stability. The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein. TopBP1 also shares many features with BRCA1. We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation. However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint. The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway. These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, we have identified the cofactor CtIP as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. We further identified the breast tumor suppressor BRCA1 as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediated interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP had no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participated in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue.

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

Lipid metabolism is essential for stemness maintenance, self‐renewal, and differentiation of stem cells, however, the regulatory function of cholesterol metabolism in erythroid differentiation is poorly studied. In the present study, a critical role for cholesterol homeostasis in terminal erythropoiesis is uncovered. The master transcriptional factor GATA1 binds to Sterol‐regulatory element binding protein 2 (SREBP2) to downregulate cholesterol biosynthesis, leading to a gradual reduction in intracellular cholesterol levels. It is further shown that reduced cholesterol functions to block erythroid proliferation via the cholesterol/mTORC1/ribosome biogenesis axis, which coordinates cell cycle exit in the late stages of erythroid differentiation. The interaction of GATA1 and SREBP2 also provides a feedback loop for regulating globin expression through the transcriptional control of NFE2 by SREBP2. Importantly, it is shown that disrupting intracellular cholesterol hemostasis resulted in defect of terminal erythroid differentiation in vivo. These findings demonstrate that fine‐tuning of cholesterol homeostasis emerges as a key mechanism for regulating erythropoiesis. [ABSTRACT FROM AUTHOR]

In keloid fibroblasts, microRNA-21 (miR-21) enhances activation of the TGF-β-Smad signaling pathway by down-regulating Smad7 expression, thereby promoting keloid fibroblast proliferation and collagen production. However, it is unclear whether miR-21 performs the above-mentioned functions through exosomal transport. Here, we extracted exosomes from the culture supernatants of keloid and normal skin fibroblasts and observed that both types of cells above secrete exosomes; however, keloid fibroblasts secreted significantly more exosomal miR-21 than normal skin fibroblasts (P < .001). Interestingly, we also observed that exosomal miR-21 could enter target keloid fibroblasts. In addition, inhibiting exosomal miR-21 up-regulated Smad7 protein expression and reduced Smad2 and Smad3 protein levels in target keloid fibroblasts. Furthermore, inhibiting exosomal miR-21 down-regulated collagen I and collagen III expression in target keloid fibroblasts, increased the proportion of apoptotic cells, and reduced cell proliferation. Taken together, these results show that exosomal miR-21 promoted proliferation and collagen production in keloid fibroblasts by inhibiting Smad7. Thus, we identified regulatory roles for miR-21 in promoting keloid fibroblast proliferation and participating in keloid formation and development. These findings imply that miR-21 may serve as a novel target for controlling the development of keloids. [ABSTRACT FROM AUTHOR]

• Surface imprinted hybrid nanofibers (FTCFs@MnO 2 @MIPs) are first developed. • Flaky MnO 2 form strong interactions with the amino groups of BSA. • FTCFs@MnO 2 @MIPs behave self-driven property. • The adsorption amount reach 816.44 mg/g within 60 min. • FTCFs@MnO 2 @MIPs exhibit excellent reusability and application ability. Along with the wide development of protein imprinted polymers, the researchers still face many challenges, such as difficult template elution, slow adsorption rate and low adsorption capacity. In order to promote the progress of protein separation and purification, the surface imprinted manganese dioxide-loaded tubular carbon fibers (FTCFs@MnO 2 @MIPs) are prepared in this work. FTCFs@MnO 2 @MIPs are based on tubular carbon fibers (TCFs) coated with flaky MnO 2. Dopamine (DA) and bovine serum albumin (BSA) are utilized as functional monomers and templates. The MnO 2 nanosheets are grown and loaded on the surface of carboxyl-modified tubular carbon fibers (CMTCFs) to form a MnO 2 shell, which provides more imprinting sites for protein imprinting. Meanwhile, this shell enhances the interaction between the imprinting sites and BSA. The content of MnO 2 loaded on the surface of CMTCFs is 9.42%. The obtained materials are systematically characterized and the adsorption performances of FTCFs@MnO 2 @MIPs for BSA are investigated. The adsorption process of FTCFs@MnO 2 @MIPs exhibits significant self-driven characteristics. The adsorption capacity reaches 816.44 mg/g in 60 min and the imprinting factor (IF) is 3.31. FTCFs@MnO 2 @MIPs can selectively separate BSA from the mixed proteins and fetal bovine serum. Excellent reusability and practical application ability make MnO 2 -loaded tubular carbon fibers (FTCFs@MnO 2) become a promising carrier in the field of protein imprinting. [ABSTRACT FROM AUTHOR]

A keloid is a benign fibroproliferative skin tumor that results from abnormal wound healing after injury and tends to grow beyond the boundary of the original wound; the mechanism of keloid formation is still unclear. MicroRNA-21 (MiR-21) is a representative microRNA that plays a key role in a variety of fibrotic diseases via the transforming growth factor-β/Smad signaling pathway. The aim of our study was to explore the mechanism of keloid formation. First, we found that the expression of miR-21 in keloids and keloid fibroblasts was significantly upregulated by microRNA microarray and real-time polymerase chain reaction. Additionally, at the protein level, our study confirmed that the overexpression of miR-21 could promote the process of keloid fibrosis to some extent and also indicated that a low expression of miR-21 could inhibit the process of keloid fibrosis. Finally, the results proved that miR-21 could participate in the keloid fibrosis process through negative regulation of its downstream target gene Smad7 via the transforming growth factor-β/Smad signaling pathway, which provides a guiding framework for further studies and new theoretical support for keloid clinical treatment. [ABSTRACT FROM AUTHOR]

The~Fanconi anemia (FA) pathway participates in interstrand crosslink (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemiaassociated proteins (FAAPZ4 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of crosslinked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP2O, which contains a RAD18like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP2O promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP2O displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP2O is an important player involved in the FA pathway. [ABSTRACT FROM AUTHOR]

Summary ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. [ABSTRACT FROM AUTHOR]

The enzyme sirtuin 1 (SIRT1) is a critical regulator of many cellular functions, including energy metabolism. However, the precise mechanisms that modulate SIRT1 activity remain unknown. As SIRT1 activity in vitro was recently found to be negatively regulated by interaction with the deleted in breast cancer-1 (DBC1) protein, we set out to investigate whether DBC1 regulates SIRT1 activity in vivo. We found that DBC1 and SIRT1 colocalized and interacted, and that DBC1 modulated SIRT1 activity, in multiple cell lines and tissues. In mouse liver, increased SIRT1 activity, concomitant with decreased DBC1-SIRT1 interaction, was detected after 24 hours of starvation, whereas decreased SIRT1 activity and increased interaction with DBC1 was observed with high-fat diet (HFD) feeding. Consistent with the hypothesis that DBC1 is crucial for HFD-induced inhibition of SIRT1 and for the development of experimental liver steatosis, genetic deletion of Dbc1 in mice led to increased SIRT1 activity in several tissues, including liver. Furthermore, DBC1-deficient mice were protected from HFD-induced liver steatosis and inflammation, despite the development of obesity. These observations define what we believe to be a new role for DBC1 as an in vivo regulator of SIRT1 activity and liver steatosis. We therefore propose that the DBC1-SIRT1 interaction may serve as a new target for therapies aimed at nonalcoholic liver steatosis. [ABSTRACT FROM AUTHOR]

Genetic instability, which leads to an accumulation of various genetic abnormalities, has been considered an essential component of the human neoplasic transformation process. However, the molecular basis of genomic instability during tumorigenesis remains incompletely understood. Growing evidence indicates that checkpoint with forkhead and ring finger domains (CHFR), a recently identified mitotic checkpoint protein, plays an important role in maintaining chromosome integrity and functions as a tumor suppressor. In this study, we used high-throughput technology to conduct gene expression profiling of human colon cancers and found that loss of CHFR expression frequently occurred in colon cancers with high microsatellite instability (MSI-H). Downregulation of CHFR expression was closely associated with overexpression of Aurora A, an important mitotic kinase. Mice with deficiencies in both Chfr and Mlh1 (the gene that encodes the DNA mismatch-repair protein Mlh1) displayed dramatically higher incidence of spontaneous tumors relative to mice deficient for only one of these genes. These results suggest that defects in both Chfr and Mlh1 synergistically increase predisposition to tumorigenesis. [ABSTRACT FROM AUTHOR]