Bidirectional coupling of splicing and ATM signaling in response to transcription-blocking DNA damage

In response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating RNA metabolism. Here we discuss a replication-independent pathway activated by transcription-blocking DNA lesions, which utilizes the ATM signaling kinase to regulate spliceosome function in a reciprocal manner. We present a model according to which, displacement of co-transcriptional spliceosomes from lesion-arrested RNA polymerases, culminates in R-loop formation and non-canonical ATM activation. ATM signals in a feed-forward fashion to further impede spliceosome organization and regulates UV-induced gene expression and alternative splicing genome-wide. This reciprocal coupling between ATM and the spliceosome highlights the importance of ATM signaling in the cellular response to transcription-blocking lesions and supports a key role of the splicing machinery in this process

FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway

Peripheral mitochondrial function correlates with clinical severity in idiopathic Parkinson’s disease

Background: Parkinson’s disease is an intractable disorder with heterogeneous clinical presentation that may reflect different underlying pathogenic mechanisms. Surrogate indicators of pathogenic processes correlating with clinical measures may assist in better patient stratification. Mitochondrial function, which is impaired in and central to PD pathogenesis, may represent one such surrogate indicator. Methods: Mitochondrial function was assessed by respirometry experiment in fibroblasts derived from idiopathic patients (n = 47) in normal conditions and in experimental settings that do not permit glycolysis and therefore force energy production through mitochondrial function. Respiratory parameters and clinical measures were correlated with bivariate analysis. Machine-learning-based classification and regression trees were used to classify patients on the basis of biochemical and clinical measures. The effects of mitochondrial respiration on α-synuclein stress were assessed monitoring the protein phosphorylation in permitting versus restrictive glycolysis conditions. Results: Bioenergetic properties in peripheral fibroblasts correlate with clinical measures in idiopathic patients, and the correlation is stronger with predominantly nondopaminergic signs. Bioenergetic analysis under metabolic stress, in which energy is produced solely by mitochondria, shows that patients’ fibroblasts can augment respiration, therefore indicating that mitochondrial defects are reversible. Forcing energy production through mitochondria, however, favors α-synuclein stress in different cellular experimental systems. Machine-learning-based classification identified different groups of patients in which increasing disease severity parallels higher mitochondrial respiration. Conclusion: The suppression of mitochondrial activity in PD may be an adaptive strategy to cope with concomitant pathogenic factors. Moreover, mitochondrial measures in fibroblasts are potential peripheral biomarkers to follow disease

Enhanced Chromatin Dynamics by FACT Promotes Transcriptional Restart after UV-Induced DNA Damage

Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response

Peripheral mitochondrial function correlates with clinical severity in idiopathic Parkinson’s disease

Background: Parkinson’s disease is an intractable disorder with heterogeneous clinical presentation that may reflect different underlying pathogenic mechanisms. Surrogate indicators of pathogenic processes correlating with clinical measures may assist in better patient stratification. Mitochondrial function, which is impaired in and central to PD pathogenesis, may represent one such surrogate indicator. Methods: Mitochondrial function was assessed by respirometry experiment in fibroblasts derived from idiopathic patients (n = 47) in normal conditions and in experimental settings that do not permit glycolysis and therefore force energy production through mitochondrial function. Respiratory parameters and clinical measures were correlated with bivariate analysis. Machine-learning-based classification and regression trees were used to classify patients on the basis of biochemical and clinical measures. The effects of mitochondrial respiration on ?-synuclein stress were assessed monitoring the protein phosphorylation in permitting versus restrictive glycolysis conditions. Results: Bioenergetic properties in peripheral fibroblasts correlate with clinical measures in idiopathic patients, and the correlation is stronger with predominantly nondopaminergic signs. Bioenergetic analysis under metabolic stress, in which energy is produced solely by mitochondria, shows that patients’ fibroblasts can augment respiration, therefore indicating that mitochondrial defects are reversible. Forcing energy production through mitochondria, however, favors ?-synuclein stress in different cellular experimental systems. Machine-learning-based classification identified different groups of patients in which increasing disease severity parallels higher mitochondrial respiration. Conclusion: The suppression of mitochondrial activity in PD may be an adaptive strategy to cope with concomitant pathogenic factors. Moreover, mitochondrial measures in fibroblasts are potential peripheral biomarkers to follow disease progression. © 2019 The Authors

Peripheral mitochondrial function correlates with clinical severity in idiopathic Parkinson’s disease

Background: Parkinson’s disease is an intractable disorder with heterogeneous clinical presentation that may reflect different underlying pathogenic mechanisms. Surrogate indicators of pathogenic processes correlating with clinical measures may assist in better patient stratification. Mitochondrial function, which is impaired in and central to PD pathogenesis, may represent one such surrogate indicator. Methods: Mitochondrial function was assessed by respirometry experiment in fibroblasts derived from idiopathic patients (n = 47) in normal conditions and in experimental settings that do not permit glycolysis and therefore force energy production through mitochondrial function. Respiratory parameters and clinical measures were correlated with bivariate analysis. Machine-learning-based classification and regression trees were used to classify patients on the basis of biochemical and clinical measures. The effects of mitochondrial respiration on ?-synuclein stress were assessed monitoring the protein phosphorylation in permitting versus restrictive glycolysis conditions. Results: Bioenergetic properties in peripheral fibroblasts correlate with clinical measures in idiopathic patients, and the correlation is stronger with predominantly nondopaminergic signs. Bioenergetic analysis under metabolic stress, in which energy is produced solely by mitochondria, shows that patients’ fibroblasts can augment respiration, therefore indicating that mitochondrial defects are reversible. Forcing energy production through mitochondria, however, favors ?-synuclein stress in different cellular experimental systems. Machine-learning-based classification identified different groups of patients in which increasing disease severity parallels higher mitochondrial respiration. Conclusion: The suppression of mitochondrial activity in PD may be an adaptive strategy to cope with concomitant pathogenic factors. Moreover, mitochondrial measures in fibroblasts are potential peripheral biomarkers to follow disease progression. © 2019 The Authors

助成研究報告

<div><p>As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) <i>Xpg^−/−</i> mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the <i>Xpg^−/−</i> mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.</p></div

Age-related increase of neuronal stress in forebrain-specific <i>Xpg</i> knockout mice.

<p>(A) Average body weight of C57Bl6/FVB F1 hybrid wild type (<i>wt</i>) females (black circles) and forebrain-specific XPG-deficient (<i>Emx1-Xpg</i>) females (gray circles); n≥4 animals/group. (B) Onset of clasping of the hind limbs in <i>Emx1-Xpg</i> mice; n = 7 animals/group. (C) Representative images of GFAP immunostained sagittal neocortex sections of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice showing progressive astrocytosis in <i>Emx1-Xpg</i> mice. (D) Representative images of Mac2 immunostained sagittal brain sections of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice showing Mac2-positive microgliosis and a progressive decrease in size of the cerebral cortex and hippocampus of <i>Emx1-Xpg</i> mice. Arrows indicate microgliosis in corpus callosum and fimbria fornix. A thionin counterstaining was used. (E) Quantification of p53-positive cells in neocortex and cerebellum of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice. Values are the average of four sections per genotype. Arrows indicate p53 positive cells. (F) Representative confocal images showing double labeled p53-NeuN cells in the neocortex (left) and p53-S100ß in the fimbria fornix (right) of 26-week old <i>Emx1-Xpg</i> mice. Arrows indicate p53 positive cells. NCx: neocortex, cc: corpus callosum, Str: striatum, ff: fimbria fornix, Hip: hippocampus. Scale bars: 50 µm (C), 500 µm (D), 200 µm (E) and 20 µm (F). Error bars indicate standard error of the mean. **p<0.01.</p

Generation of <i>Xpg^−/−</i> mice.

<p>(A) Genomic organization and disruption strategy for <i>Xpg</i> depicting the wild type allele (<i>+</i>), the targeting construct, the targeted allele (<i>fn</i>), the conditional allele after Flp-mediated recombination of <i>Frt</i> sites (<i>f</i>) and the targeted <i>Xpg</i> allele following subsequent Cre-mediated recombination of <i>LoxP</i> sites (<i>−</i>). Exons 2–5 are indicated by black boxes. PCR primers are shown as arrows. (B) Southern blot and PCR analysis of an ES clone showing the correct insertion of the targeting construct. ES cell genomic DNA was digested with EcoRI for Southern blot analysis and hybridized with a 0.9 kb DpnI probe. The wild type (<i>wt</i>) allele yields a 7.4-kb fragment whereas the targeted (<i>tg</i>) allele yields a 4.1-kb fragment. The NheI-digested PCR product shows the 2.3-kb and 2.2-kb bands corresponding with the <i>wt</i> and <i>tg</i> allele, respectively (see also panel A). (C) PCR detection of mouse genotypes using the primers F1, NeoF and R1 as indicated as in A. (D) Immunoblot analysis of extracts from <i>Xpg^−/−</i> and <i>wt</i> MDFs using a rabbit polyclonal antibody raised against a peptide conserved between human and mouse XPG. Tubulin is used as loading control. (E) Primary <i>Xpg^−/−</i> and <i>wt</i> MDFs, cultured at low (3%) O₂ levels were irradiated with the indicated doses of UV-C (left) or treated with the indicated doses of Illudin S for 1 h (right). After 48 h recovery, survival was assessed by cell count. (F) UV-induced UDS in primary <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe GG-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. UDS levels are expressed relative to the non-irradiated <i>wt</i> cells. (G) UV-induced RRS in primary <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe TC-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. 16 h after UV irradiation the <i>wt</i> cells show recovery of RNA synthesis, while <i>Xpg^−/−</i> MDFs only show residual activity in nucleoli (rRNA transcription). Arrowheads indicate nuclei. Error bars indicate standard error of the mean. **p<0.01.</p