Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA-binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Re-assigning the histologic identities of COV434 and TOV-112D ovarian cancer cell lines.

OBJECTIVE: The development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors. METHODS: For COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical follow-up on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells. RESULTS: The available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically. CONCLUSIONS: COV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.

Clinical utility of SMARCA4 testing by immunohistochemistry in rare ovarian tumours.

BACKGROUND: Ovarian small cell carcinoma, hypercalcaemic type (SCCOHT) is a rare and lethal disease affecting young women. As histological diagnosis is challenging and urgent, there is a clear need for a robust diagnostic test. While mutations in the chromatin-remodelling gene, SMARCA4, appear to be typical, it may not be feasible routinely to be clinically relevant. METHODS: Previous studies have described the value of SMARCA4 IHC to differentiate SCCOHT from ovarian neoplasms (ON), with similar histologic appearances. We aimed to evaluate its clinical utility among a cohort of 44 SCCOHT and 94 rare ON frequently misdiagnosed as SCCOHT. RESULTS: Forty-three percent (16/36) of SCCOHT had been classified locally as non-SCCOHT confirming the diagnosis challenge. Sensitivity and specificity of SMARCA4 IHC were excellent at 88% and 94%, respectively. In a community setting with a much lower prevalence of the disease, estimated PPV is 40% while NPV remained high at 99%. Finally, among the 16 SCCOHT misclassified locally, SMARCA4 IHC testing would have resulted in corrected diagnosis in 88% of cases. CONCLUSIONS: SMARCA4 IHC is a highly sensitive, and specific test for the diagnosis of SCCOHT and is of huge clinical utility in providing a timely and accurate diagnosis of this challenging disease.

Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability.

A large number of SNF2 family, DNA and ATP-dependent motor proteins are needed during transcription, DNA replication, and DNA repair to manipulate protein-DNA interactions and change DNA structure. SMARCAL1, ZRANB3, and HLTF are three related members of this family with specialized functions that maintain genome stability during DNA replication. These proteins are recruited to replication forks through protein-protein interactions and bind DNA using both their motor and substrate recognition domains (SRDs). The SRD provides specificity to DNA structures like forks and junctions and confers DNA remodeling activity to the motor domains. Remodeling reactions include fork reversal and branch migration to promote fork stabilization, template switching, and repair. Regulation ensures these powerful activities remain controlled and restricted to damaged replication forks. Inherited mutations in SMARCAL1 cause a severe developmental disorder and mutations in ZRANB3 and HLTF are linked to cancer illustrating the importance of these enzymes in ensuring complete and accurate DNA replication. In this review, we examine how these proteins function, concentrating on their common and unique attributes and regulatory mechanisms.

Identification of Helicase Proteins as Clients for HSP90.

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that maintains the proper folding of its client proteins including protein kinases and steroid hormone receptors. Helicases are a group of nucleic acid-binding ATPases that can unwind DNA and/or RNA and function in almost every aspect of nucleic acid metabolism. Not much, however, is known about the interactions between HSP90 and helicase proteins. Herein, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method that allows for quantifying >80% of the human helicase proteome. By employing this method, we demonstrated that a large number of helicase proteins exhibited diminished expression in cultured human cells upon treatment with two small-molecule inhibitors of HSP90. We further introduced a tandem affinity tag to the C-terminus of endogenous HSP90ß protein by using the CRISPR-Cas9 genome editing method. Affinity purification followed by LC-PRM analysis revealed an enrichment of 40 out of the 66 quantified helicases from the lysate of cells expressing tagged HSP90ß. Together, we developed a high-throughput targeted proteomic method for assessing quantitatively the human helicase proteome, and our results support that helicases may constitute an important group of client proteins for HSP90.

SMARCA4 inactivating mutations cause concomitant Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary hypercalcaemic type.

SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mutation in TDRD9 causes non-obstructive azoospermia in infertile men.

BACKGROUND: Azoospermia is diagnosed when sperm cells are completely absent in the ejaculate even after centrifugation. It is identified in approximately 1% of all men and in 10%-20% of infertile males. Non-obstructive azoospermia (NOA) is characterised by the absence of sperm due to either a Sertoli cell-only pattern, maturation arrest, hypospermatogenesis or mixed patterns. NOA is a severe form of male infertility, with limited treatment options and low fertility success rates. In the majority of patients, the cause for NOA is not known and mutations in only a few genes were shown to be causative. AIM: We investigated the cause of maturation arrest in five azoospermic infertile men of a large consanguineous Bedouin family. METHODS AND RESULTS: Using whole genome genotyping and exome sequencing we identified a 4 bp deletion frameshift mutation in TDRD9 as the causative mutation with a Lod Score of 3.42. We demonstrate that the mutation results in a frameshift as well as exon skipping. Immunofluorescent staining with anti-TDRD9 antibody directed towards the N terminus demonstrated the presence of the protein in testicular biopsies of patients with an intracellular distribution comparable to a control biopsy. The mutation does not cause female infertility. CONCLUSION: This is the first report of a recessive deleterious mutation in TDRD9 in humans. The clinical phenotype recapitulates that observed in the Tdrd9 knockout mice where this gene was demonstrated to participate in long interspersed element-1 retrotransposon silencing. If this function is preserved in human, our data underscore the importance of maintaining DNA stability in the human male germ line.

SMARCA4-deficient thoracic sarcoma: a distinctive clinicopathological entity with undifferentiated rhabdoid morphology and aggressive behavior.

A distinct subset of thoracic sarcomas with undifferentiated rhabdoid morphology and SMARCA4 inactivation has recently been described, and potential targeted therapy for SMARC-deficient tumors is emerging. We sought to validate the clinicopathological features of SMARCA4-deficient thoracic sarcomas. Clinicopathological information was gathered for 40 undifferentiated thoracic tumors with rhabdoid morphology (mediastinum (n=18), lung (n=14), pleura (n=8)). Thymic carcinomas (n=11) were used as a comparison group. Immunohistochemistry included BRG1 (SMARCA4), BRM (SMARCA2), INI-1 (SMARCB1), pan-cytokeratin, desmin, NUT, S-100 protein, TTF1, CD34, and SOX2. BRG1 loss was present in 12 of 40 rhabdoid thoracic tumors (30%): 7 of 18 in mediastinum (39%), 2 of 8 in pleura (25%), and 3 of 14 in lung (21%). All BRG1-deficient tumors tested for BRM (n=8) showed concomitant loss. All thymic carcinomas showed retained BRG1 and INI-1. Morphologically, tumors with BRG1 loss showed sheets of monotonous ovoid cells with indistinct cell borders, abundant eosinophilic cytoplasm, and prominent nucleoli. Scattered areas with rhabdoid morphology (ie, eccentric nuclei, dense eosinophilic cytoplasm, discohesion) were present in all the cases. SMARCA4/BRG1-deficient sarcomas showed rare cells positive for cytokeratin in 10 cases (83%). One showed rare TTF1-positive cells. All were negative for desmin, NUT, and S-100 protein. CD34 was positive in three of five (60%) BRG1-deficient tumors tested. SOX2 was positive in all four BRG1-deficient tumors tested, and negative in all seven tested cases with retained BRG1. SMARCA4/BRG1-deficient sarcomas occurred at median age of 59 years (range 44-76) with male predominance (9:3) and had worse 2-year survival compared with BRG1-retained tumors (12.5% vs 64.4%, P=0.02). SMARCA4-deficient thoracic sarcomas can be identified based on their distinctive high-grade rhabdoid morphology, and the diagnosis can be confirmed by immunohistochemistry. Identification of these tumors is clinically relevant due to their aggressive behavior, poor prognosis, and potential targeted therapy.

Loss of expression of SMARCA4 (BRG1), SMARCA2 (BRM) and SMARCB1 (INI1) in undifferentiated carcinoma of the endometrium is not uncommon and is not always associated with rhabdoid morphology.

AIM: Abnormalities of SMARCB1 (INI1), which encodes a member of the SWI/SNF pathway, are found in neoplasms with rhabdoid morphology, such as malignant rhabdoid tumour of the kidney and atypical teratoid/rhabdoid tumour of the central nervous system. SMARCA4 (BRG1), which encodes another member of the SWI/SNF pathway, and which is mutated in almost all small-cell carcinomas of the ovary, hypercalcaemic type, has been investigated in endometrial carcinomas, and mutations with resultant loss of immunohistochemical staining have been demonstrated in some endometrial undifferentiated carcinomas/dedifferentiated carcinomas. The aim of this study was to evaluate immunohistochemical expression of SMARCA4, SMARCB1 and SMARCA2 in a cohort of undifferentiated endometrial carcinomas, and to correlate expression of these markers with rhabdoid morphology and clinical outcome. METHODS AND RESULTS: Forty undifferentiated endometrial carcinomas (18 pure and 22 dedifferentiated carcinomas) were stained with SMARCA4 (n = 40), SMARCB1 (n = 27), and SMARCA2 (n = 37). SMARCA4 expression was intact in 26 of 40 (65%) cases, lost in 13 of 40 (32.5%) cases, and unassessable in one case (2.5%). SMARCB1 expression was intact in 26 of 27 (96%) cases and lost in one of 27 (4%) cases. SMARCA2 expression was intact in 23 of 37 (62%) cases, lost in 10 of 37 (27%) cases, and unassessable in four cases. SMARCA2 expression showed corresponding loss in nine of the 13 (69%) SMARCA4-deficient cases. Rhabdoid morphology was present in three of 13 (23%) SMARCA4-deficient cases, in two of 10 (20%) SMARCA2-deficient cases, in four of 26 (15%) SMARCA4-intact cases, and in four of 23 (17%) SMARCA2-intact cases. There was no correlation between SMARCA4 or SMARCA2 expression and clinical outcome. CONCLUSIONS: Our study demonstrated that almost one-third of endometrial undifferentiated carcinomas show loss of SMARCA4 and SMARCA2 expression, and that a subset show rhabdoid morphology. The majority of the SMARCA4-deficient cases show concomitant loss of SMARCA2 expression. There is no correlation between SMARCA4 or SMARCA2 expression and outcome. Our results confirm that the SWI/SNF chromatin-remodelling complex is involved in the pathogenesis of endometrial undifferentiated carcinomas.

RUVBL1-ITFG1 interaction is required for collective invasion in breast cancer.

BACKGROUND: The mechanisms of breast cancer collective invasion are poorly understood limiting the metastasis therapy. The ATPase RUVBL1 is frequently overexpressed in various cancers and plays a crucial role in oncogenic process. We further investigated the role of RUVBL1 in promoting collective invasion and uncovered that targeting RUVBL1 could inhibit metastatic progression. METHODS: The expression levels of RUVBL1 and ITFG1 were examined by Western blot and qRT-PCR. Co-localization and interaction of RUVBL1 and ITFG1 were determined by immunofluorescence and co-immunoprecipitation. The invasive ability was examined by transwell assay and microfluidic assay. The metastatic and tumorigenic abilities of breast cancer cells were revealed in BALB/c nude mice by xenograft and tail vein injection. RESULTS: ATPase RUVBL1 is highly expressed in breast cancer and predicts the poor prognosis. Elevated expression of RUVBL1 is found in high metastatic breast cancer cells. Silencing RUVBL1 suppresses cancer cell expansion and invasion in vitro and in vivo. RUVBL1 interacts with a conserved transmembrane protein ITFG1 in cytoplasm and plasma membrane to promote the collective invasion. Using a microfluidic model, we demonstrated that silencing RUVBL1 or ITFG1 individually compromises collective invasion of breast cancer cells. CONCLUSION: RUVBL1 is a vital regulator for collective invasion. The interaction between RUVBL1 and ITFG1 is required for breast cancer cell collective invasion and progression. GENERAL SIGNIFICANCE: Targeting collective invasion promoted by RUVBL1-ITFG1 complex provides a novel therapeutic strategy to improve the prognosis of invasive breast cancer.

Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy.

A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM).

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

[Oridonin inhibits proliferation of Jurkat cells via the down-regulation of Brg1].

OBJECTIVE: To investigate the effect of oridonin on the human acute lymphocytic leukemia cell line Jurkat and its mechanism. METHODS: Jurkat cells were cultured in vitro and treated with various concentrations (0, 1.25, 2.5, 5, and 10 µmol/L) of oridonin for different lengths of time (24, 48, and 72 hours). The proliferation of Jurkat cells was analyzed by MTT assay. The changes in nuclear morphology were evaluated by fluorescence microscopy at 12 hours after treatment with various concentrations of oridonin. The expression levels of Brg1, P53, and C-myc were determined by semi-quantitative Western blot in Jurkat cells treated with various concentrations of oridonin for 24 hours or 5 µmol/L oridonin for various lengths of time (0, 2, 6, 12, and 24 hours). The expression levels of P53 and C-myc and proliferation of Jurkat cells were evaluated after Brg1 expression was knocked down by Brg1-specific siRNA. RESULTS: Compared with the control group, the proliferation of oridonin-treated Jurkat cells was significantly inhibited in a concentration- and time-dependent manner (P<0.05). According to the florescence microscopic analysis, oridonin treatment led to nuclear pyknosis in Jurkat cells. Compared with the control group, Jurkat cells treated with 5 µmol/L oridonin had reduced expression of Brg1 and C-myc but elevated expression of P53. Brg1 knock-down led to a significant reduction in proliferation of Jurkat cells (P<0.05), up-regulated expression of P53, and down-regulated expression of C-myc. CONCLUSIONS: Oridonin can inhibit the proliferation of Jurkat cells, probably via the Brg1 signaling pathway.

Novel subcellular localization of the DNA helicase Twinkle at the kinetochore complex during mitosis in neuronal-like progenitor cells.

During mitosis, the kinetochore, a multi-protein structure located on the centromeric DNA, is responsible for proper segregation of the replicated genome. More specifically, the outer kinetochore complex component Ndc80/Hec1 plays a critical role in regulating microtubule attachment to the spindle for accurate sister chromatid segregation. In addition, DNA helicases play a key contribution for precise and complete disjunction of sister chromatids held together through double-stranded DNA catenations until anaphase. In this study, we focused our attention on the nuclear-encoded DNA helicase Twinkle, which functions as an essential helicase for replication of mitochondrial DNA. It regulates the copy number of the mitochondrial genome, while maintaining its integrity, two processes essential for mitochondrial biogenesis and bioenergetic functions. Although the majority of the Twinkle protein is imported into mitochondria, a small fraction remains cytosolic with an unknown function. In this study, we report a novel expression pattern of Twinkle during chromosomal segregation at distinct mitotic phases. By immunofluorescence microscopy, we found that Twinkle protein colocalizes with the outer kinetochore protein HEC1 as early as prophase until late anaphase in neuronal-like progenitor cells. Thus, our collective results have revealed an unexpected cell cycle-regulated expression pattern of the DNA helicase Twinkle, known for its role in mtDNA replication. Therefore, its recruitment to the kinetochore suggests an evolutionary conserved function for both mitochondrial and nuclear genomic inheritance.

Loss of SMARCA4 (BRG1) protein expression as determined by immunohistochemistry in small-cell carcinoma of the ovary, hypercalcaemic type distinguishes these tumours from their mimics.

AIMS: Molecular investigation of small-cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) has revealed that it is a monogenetic tumour characterized by alteration of SMARCA4 (BRG1), encoding a member of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex. A large majority of cases show loss of expression of the corresponding SMARCA4/BRG1 protein. Furthermore, three cases of SCCOHT with retained SMARCA4 protein expression showed loss of SMARCB1/INI1 expression. The aim of this study was to assess the sensitivity and specificity of loss of SMARCA4 expression as a diagnostic test for SCCOHT. METHODS AND RESULTS: We performed SMARCA4 and SMARCB1 staining in 245 tumours, many of which were potentially in the differential diagnosis of SCCOHT. We also stained 56 cases of SCCOHT for SMARCA4 and 37 of these for SMARCB1. Fifty-four of the SCCOHT cases showed complete absence of SMARCA4 expression. The two cases with retained expression showed molecular alteration of SMARCA4. Of the 217 other neoplasms with interpretable staining, all retained SMARCA4 expression. Although the majority showed diffuse, strong nuclear expression, a heterogeneous, typically weak staining pattern was present in 13% of cases. All 37 cases of SCCOHT tested and all other neoplasms, apart from three malignant rhabdoid tumours, showed retained nuclear SMARCB1 expression. Loss of SMARCA4 expression had a sensitivity of 96.55% and specificity of 100%. CONCLUSIONS: Loss of SMARCA4 expression is sensitive and specific for SCCOHT. Although some mimics show heterogeneous expression, there is retention of nuclear staining in at least a part of the tumour; therefore, only complete loss of staining should be regarded as being supportive of SCCOHT.

Study of clinicopathological features, hormone immunoexpression, and loss of ATRX and DAXX expression in pancreatic neuroendocrine tumors.

OBJECTIVES: Neuroendocrine tumors of the pancreas (PanNETs) are rare neoplasms, and not much is known about their pathogenesis. We aimed to evaluate ATRX/DAXX immunoexpression in PanNETs a cohort of well-characterized PanNETs. METHODS: PanNETs diagnosed over a 10-year period were retrieved and clinicopathogical features reviewed. Immuohistochemistry for pancreatic hormones, and for ATRX and DAXX was performed. RESULTS: Sixty-eight PanNETs were included (30 males and 38 females) with median age of 39 years. Histologically, there were 37 Grade 1 (54.4%), 27 Grade 2 (39.7%), and 4 Grade 3 (5.9%) cases. On immunostaining for hormones, insulin expression was most frequent (22 cases; 38.6%), followed by gastrin (7 cases; 12.3%); 25 cases (43.9%) were negative for all hormones. Loss of ATRX/DAXX immunoexpression was noted in 18 cases (39.1%), and was significantly more frequent in tumors larger than 5 cm. Lymphovascular invasion, infiltrative borders, and infiltration of adjacent organs were also more frequent in tumors with loss of ATRX/DAXX immunoreactivity. A little over half the tumors with ATRX/DAXX loss showed negative immunostaining for all hormones (55.6%). CONCLUSION: Loss of ATRX/DAXX expression is frequent in PanNETs, indicating a role in their pathogenesis. As ATRX/DAXX loss is more frequent in larger tumors, and in those with lymphovascular invasion, adjacent organ infiltration and infiltrative borders, this suggests that loss of ATRX/DAXX expression is a late event in pathogenesis and is associated with an aggressive phenotype. Immunohistochemical detection of ATRX/DAXX loss is a simple method for ATRX/DAXX evaluation and can easily be incorporated into routine pathological evaluation of PanNETs.

Genome-wide reorganization of histone H2AX toward particular fragile sites on cell activation.

γH2AX formation by phosphorylation of the histone variant H2AX is the key process in the repair of DNA lesions including those arising at fragile sites under replication stress. Here we demonstrate that H2AX is dynamically reorganized to preoccupy γH2AX hotspots on increased replication stress by activated cell proliferation and that H2AX is enriched in aphidicolin-induced replisome stalling sites in cycling cells. Interestingly, H2AX enrichment was particularly found in genomic regions that replicate in early S phase. High transcription activity, a hallmark of early replicating fragile sites, was a determinant of H2AX localization. Subtelomeric H2AX enrichment was also attributable to early replication and high gene density. In contrast, late replicating and infrequently transcribed regions, including common fragile sites and heterochromatin, lacked H2AX enrichment. In particular, heterochromatin was inaccessible to H2AX incorporation, maybe partly explaining the cause of mutation accumulation in cancer heterochromatin. Meanwhile, H2AX in actively dividing cells was intimately colocalized with INO80. INO80 silencing reduced H2AX levels, particularly at the INO80-enriched sites. Our findings suggest that active DNA replication is accompanied with the specific localization of H2AX and INO80 for efficient damage repair or replication-fork stabilization in actively transcribed regions.

Length-dependent processing of telomeres in the absence of telomerase.

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5'-3' resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5'-3' resection at the shortest telomeres.

Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication.

Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.