Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

Acknowledgements We thank all members of the Di Virgilio lab for their feedback and discussion; V Delgado-Benito (Di Virgilio lab, MDC, Berlin) for her contribution to the project development; L Keller (Di Virgilio lab, MDC, Berlin) for support with cloning, mutagenesis, and mice genotyping; C Brischetto (Scheidereit Lab, MDC, Berlin) for assistance with confocal microscopy; Aberdeen Proteomics facility (University of Aberdeen) for the mass spec analysis of Aph-induced hRIF1 phosphorylation; and the MDC FACS Core Facility and Dr. HP Rahn for support with cell sorting. Aliquots of ATRi and ATMi were gener- ously provided by AG Henssen (MDC and ECRC, Berlin). Figures 1B and D, 2A, and 4C contain items created with BioRender.com. This work was supported by ERC grant 638897 (to MDV), the Helmholtz- Gemeinschaft Zukunftsthema 'Immunology and Inflammation' ZT-0027 (to MDV), P41 GM109824 and P41 GM103314 (to BTC), and Cancer Research UK awards C1445/A19059 and DRCPGM\100,013 (to ADD and SH).Peer reviewe

Silymarin Inhibits Cytokine-Stimulated Pancreatic Beta Cells by Blocking the ERK1/2 Pathway

We show that silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), inhibits cytokine mixture (CM: TNF-α, IFN-γ, and IL-1β)-induced production of nitric oxide (NO) in the pancreatic beta cell line MIN6N8a. Immunostaining and Western blot analysis showed that silymarin inhibits iNOS gene expression. RT-PCR showed that silymarin inhibits iNOS gene expression in a dose-dependent manner. We also showed that silymarin inhibits extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) phosphorylation. A MEK1 inhibitor abrogated CM-induced nitrite production, similar to silymarin. Treatment of MIN6N8a cells with silymarin also inhibited CM-stimulated activation of NF-κB, which is important for iNOS transcription. Collectively, we demonstrate that silymarin inhibits NO production in pancreatic beta cells, and silymarin may represent a useful anti-diabetic agent

miR146a-mediated Targeting of FANCM during Inflammation Compromises Genome Integrity

Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3’ untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM

miR146a-mediated Targeting of FANCM during Inflammation Compromises Genome Integrity

Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3’ untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM

miR146a-Mediated Targeting of FANCM during Inflammation Compromises Genome Integrity

Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3’ untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM

Disintegration of the NuRD Complex in Primary Human Muscle Stem Cells in Critical Illness Myopathy

Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.</jats:p

Pdap1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification

AbstractThe establishment of protective humoral immunity is dependent on the ability of mature B cells to undergo antibody gene diversification while adjusting to the physiological stressors induced by activation with the antigen. Mature B cells diversify their antibody genes by class switch recombination (CSR) and somatic hypermutation (SHM), which are both dependent on efficient induction of activation-induced cytidine deaminase (AID). Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation and ensures efficient antibody diversification by promoting their survival and optimal function.</jats:p