BL09XU: an advanced hard X-ray photoelectron spectroscopy beamline of SPring-8.

The BL09XU beamline of SPring-8 has been reorganized into a beamline dedicated for hard X-ray photoelectron spectroscopy (HAXPES) to provide advanced capabilities with upgraded optical instruments. The beamline has two HAXPES analyzers to cover a wide range of applications. Two sets of double channel-cut crystal monochromators with the Si(220) and (311) reflections were installed to perform resonant HAXPES analyses with a total energy resolution of less than 300 meV over a wide energy range (4.9-12 keV) while achieving a fixed-exit condition. A double-crystal X-ray phase retarder using diamond crystals controls the polarization state with a high degree of polarization over 0.9 in the wide energy range 5.9-9.5 keV. Each HAXPES analyzer is equipped with a focusing mirror to provide a high-flux microbeam. The design and performance of the upgraded instruments are presented.

Transcriptional elongation factor ENL phosphorylated by ATM recruits polycomb and switches off transcription for DSB repair.

Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity.

The BRCA1/BARD1-interacting protein OLA1 functions in centrosome regulation.

The breast and ovarian cancer-specific tumor suppressor BRCA1, along with its heterodimer partner BRCA1-associated RING domain protein (BARD1), plays important roles in DNA repair, centrosome regulation, and transcription. To explore further functions of BRCA1/BARD1, we performed mass spectrometry analysis and identified Obg-like ATPase 1 (OLA1) as a protein that interacts with the carboxy-terminal region of BARD1. OLA1 directly bound to the amino-terminal region of BRCA1 and γ-tubulin. OLA1 localized to centrosomes in interphase and to the spindle pole in mitotic phase, and its knockdown resulted in centrosome amplification and the activation of microtubule aster formation. OLA1 with a mutation observed in breast cancer cell line, E168Q, failed to bind BRCA1 and rescue the OLA1 knockdown-induced centrosome amplification. BRCA1 variant I42V also abrogated the binding of BRCA1 to OLA1. These findings suggest that OLA1 plays an important role in centrosome regulation together with BRCA1.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

Purpose: Spermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1. Methods: To elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry. Result: The Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid-pachytene spermatocytes to the early spermatids. Conclusion: Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non-obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

Multilateral surface analysis of the CeB6 electron-gun cathode used at SACLA XFEL.

The CeB6(001) single crystal used as a cathode in a low-emittance electron gun and operated at the free-electron laser facility SACLA was investigated using cathode lens electron microscopy combined with X-ray spectroscopy at SPring-8 synchrotron radiation facility. Multilateral analysis using thermionic emission electron microscopy, low-energy electron microscopy, ultraviolet and X-ray photoemission electron microscopy and hard X-ray photoemission spectroscopy revealed that the thermionic electrons are emitted strongly and evenly from the CeB6 surface after pre-activation treatment (annealing at 1500°C for >1 h) and that the thermionic emission intensity as well as elemental composition vary between the central area and the edge of the old CeB6 surface.

AMPK Complex Activation Promotes Sarcolemmal Repair in Dysferlinopathy.

Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.

Relationship among DNA double-strand break (DSB), DSB repair, and transcription prevents genome instability and cancer.

DNA double-strand break (DSB) is a serious type of DNA damage and is known to trigger multiple responses within cells. In these responses, novel relationships among DSB, DSB repair, and transcription machineries are created. First, transcription is repressed if DSB occurs near or at the transcription site, termed DSB-induced transcriptional repression, which contributes to DSB repair with the aid of DNA damage-signaling pathways, ATM- or DNA-PKcs-signaling pathways. DSB-induced transcriptional repression is also regulated by transcriptional factors TLP1, NELF, and ENL, as well as chromatin remodeling and organizing factors ZMYND8, CDYL1, PBAF, and cohesin. Second, transcription and RNA promote DSB repair for genome integrity. Transcription factors such as LEDGF, SETD2, and transcriptionally active histone modification, H3K36, facilitate homologous recombination to overcome DSB. At transcriptional active sites, DNA:RNA hybrids, termed R-loops, which are formed by DSB, are processed by RAD52 and XPG leading to an activation of the homologous recombination pathway. Even in a transcriptionally inactive non-genic sites, noncoding RNAs that are produced by RNA polymerase II, DICER, and DROSHA, help to recruit DSB repair proteins at the DSB sites. Third, transcriptional activation itself, however, can induce DSB. Transcriptional activation often generates specific DNA structures such as R-loops and topoisomerase-induced DSBs, which cause genotoxic stress and may lead to genome instability and consequently to cancer. Thus, transcription and DSB repair machineries interact and cooperate to prevent genome instability and cancer.

The hMsh2-hMsh6 complex acts in concert with monoubiquitinated PCNA and Pol η in response to oxidative DNA damage in human cells.

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.

Realization of a scanning soft X-ray microscope for magnetic imaging under high magnetic fields.

For the purpose of imaging element- and shell-specific magnetic distributions under high magnetic fields, a scanning soft X-ray microscope has been developed at beamline BL25SU, SPring-8, Japan. The scanning X-ray microscope utilizes total electron yield detection of absorbed circularly polarized soft X-rays in order to observe magnetic domains through the X-ray magnetic circular dichroism effect. Crucially, this system is equipped with an 8 T superconducting magnet. The performance and features of the present system are demonstrated by magnetic domain observations of the fractured surface of a Nd14.0Fe79.7Cu0.1B6.2 sintered magnet.

The ACF1 complex is required for DNA double-strand break repair in human cells.

DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.

Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin.

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼ 90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Insulin receptor substrate-4 binds to Slingshot-1 phosphatase and promotes cofilin dephosphorylation.

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment.

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.

Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose).

Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.

Polycomb group protein PHF1 regulates p53-dependent cell growth arrest and apoptosis.

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression.

Curcumin suppresses multiple DNA damage response pathways and has potency as a sensitizer to PARP inhibitor.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are promising anticancer drugs, particularly for the treatment of tumors deficient in the DNA damage response (DDR). However, it is challenging to design effective therapeutic strategies for use of these compounds against cancers without DDR deficiencies. In this context, combination therapies in which PARP inhibitors are used alongside DDR inhibitors have elicited a great deal of interest. Curcumin, a component of turmeric (Curcuma longa), has been tested in clinical studies for its chemosensitizing potential; however, the mechanisms of chemosensitization by curcumin have not been fully elucidated. This study demonstrates that curcumin suppresses three major DDR pathways: non-homologous end joining (NHEJ), homologous recombination (HR) and the DNA damage checkpoint. Curcumin suppresses the histone acetylation at DNA double-strand break (DSB) sites by inhibiting histone acetyltransferase activity, thereby reducing recruitment of the key NHEJ factor KU70/KU80 to DSB sites. Curcumin also suppresses HR by reducing expression of the BRCA1 gene, which regulates HR, by impairing histone acetylation at the BRCA1 promoter. Curcumin also inhibits ataxia telangiectasia and Rad3-related protein (ATR) kinase (IC50 in vitro = 493 nM), resulting in impaired activation of ATR-CHK1 signaling, which is necessary for HR and the DNA damage checkpoint pathway. Thus, curcumin suppresses three DDR pathways by inhibiting histone acetyltransferases and ATR. Concordantly, curcumin sensitizes cancer cells to PARP inhibitors by enhancing apoptosis and mitotic catastrophe via inhibition of both the DNA damage checkpoint and DSB repair. Our results indicate that curcumin is a promising sensitizer for PARP inhibitor-based therapy.

Nucleoporin Nup188 is required for chromosome alignment in mitosis.

Most cancer cells are aneuploid, which could be caused by defects in chromosome segregation machinery. Nucleoporins (Nup) are components of the nuclear pore complex, which is essential for nuclear transport during interphase, but several nucleoporins are also known to be involved in chromosome segregation. Here we report a novel function of Nup188, one of the nucleoporins regulating chromosome segregation. Nup188 localizes to spindle poles during mitosis, through the C-terminal region of Nup188. In Nup188-depleted mitotic cells, chromosomes fail to align to the metaphase plate, which causes mitotic arrest due to the spindle assembly checkpoint. Both the middle and the C-terminal regions were required for chromosome alignment. Robust K-fibers, microtubule bundles attaching to kinetochores, were hardly formed in Nup188-depleted cells. Significantly, we found that Nup188 interacts with NuMA, which plays an instrumental role in focusing microtubules at centrosomes, and NuMA localization to spindle poles is perturbed in Nup188-depleted cells. These data suggest that Nup188 promotes chromosome alignment through K-fiber formation and recruitment of NuMA to spindle poles.

A transfection reporter for the prevention of false-negative results in molecular beacon experiments.

We previously developed a molecular beacon-type probe to detect the strand scission in cellular base excision repair and found that the phosphodiester linkages in the fluorophore/quencher linkers were cleaved. This reaction was applied to a transfection reporter, which contained the unmodified phosphodiester in the linker to another type of fluorophore. After cotransfection of cells with the probe and the reporter, the signals were used to detect the incision and to confirm the proper transfection, respectively. This method will contribute to the prevention of false-negative results in experiments using molecular beacon-type probes.

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3' exonuclease and can participate in DNA end joining in a biochemical system.

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF, also called aprataxin- and PNK-like factor (APLF)) has been shown to have nuclease activity and to use its forkhead-associated domain to bind to x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4). Because XRCC4 is a key component of the ligase IV complex that is central to the nonhomologous DNA end joining (NHEJ) pathway, this raises the possibility that PALF might play a role in NHEJ. For this reason, we further studied the nucleolytic properties of PALF, and we searched for any modulation of PALF by NHEJ components. We verified that PALF has 3' exonuclease activity. However, PALF also possesses single-stranded DNA endonuclease activity. This single-stranded DNA endonuclease activity can act at all single-stranded sites except those within four nucleotides 3' of a double-stranded DNA junction, suggesting that PALF minimally requires approximately four nucleotides of single-strandedness. Ku, DNA-dependent protein kinase catalytic subunit, and XRCC4-DNA ligase IV do not modulate PALF nuclease activity on single-stranded DNA or overhangs of duplex substrates. PALF does not open DNA hairpins. However, in a reconstituted end joining assay that includes Ku, XRCC4-DNA ligase IV, and PALF, PALF is able to resect 3' overhanging nucleotides and permit XRCC4-DNA ligase IV to complete the joining process in a manner that is as efficient as Artemis. Reduction of PALF in vivo reduces the joining of incompatible DNA ends. Hence, PALF can function in concert with other NHEJ proteins.

Human DNA polymerase iota protects cells against oxidative stress.

Human DNA polymerase iota (poliota) is a unique member of the Y-family of specialised polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although poliota is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biological importance of poliota in human cells, we generated fibroblasts stably downregulating poliota (MRC5-pol iota(KD)) and examined their response to several types of DNA-damaging agents. We show that cell lines downregulating poliota exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H(2)O(2)) or menadione but not to ethylmethane sulphonate (EMS), UVC or UVA. Interestingly, extracts from cells downregulating poliota show reduced base excision repair (BER) activity. In addition, poliota binds to chromatin after treatment of cells with H(2)O(2) and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged poliota accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human poliota in protecting cells from oxidative damage.