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 reviewedPublisher PD

An Integrative Computational Approach for Prioritization of Genomic Variants

An essential step in the discovery of molecular mechanisms contributing to disease phenotypes and efficient experimental planning is the development of weighted hypotheses that estimate the functional effects of sequence variants discovered by high-throughput genomics. With the increasing specialization of the bioinformatics resources, creating analytical workflows that seamlessly integrate data and bioinformatics tools developed by multiple groups becomes inevitable. Here we present a case study of a use of the distributed analytical environment integrating four complementary specialized resources, namely the Lynx platform, VISTA RViewer, the Developmental Brain Disorders Database (DBDB), and the RaptorX server, for the identification of high-confidence candidate genes contributing to pathogenesis of spina bifida. The analysis resulted in prediction and validation of deleterious mutations in the SLC19A placental transporter in mothers of the affected children that causes narrowing of the outlet channel and therefore leads to the reduced folate permeation rate. The described approach also enabled correct identification of several genes, previously shown to contribute to pathogenesis of spina bifida, and suggestion of additional genes for experimental validations. The study demonstrates that the seamless integration of bioinformatics resources enables fast and efficient prioritization and characterization of genomic factors and molecular networks contributing to the phenotypes of interest.</p

Entschlüsselung des Mechanismus von RIF1 zur Aufrechterhaltung der DNA-Replikations-assoziierten Genomstabilität

Timely and accurate genome duplication is essential to maintain genome integrity and cell survival. DNA replication-associated damage is one of the leading causes of genome instability and a precursor for carcinogenesis. The DNA replication fork (RF), the site for assembly of replication proteins, encounters a variety of obstacles, which slow or stall its progression, a process termed replication stress. Cells have evolved a number of mechanisms to stabilize stalled forks and to ensure replication restart and timely completion. However, during chronic stress, forks can no longer be stabilized and collapse, creating toxic DNA double-strand breaks (DSB). These DSBs, when left unrepaired, can lead to chromosomal rearrangements and promote genomic instability. RIF1, a multifunctional protein, is critical not only to promote fork stability and to ensure that replication is completed, but also to repair DSBs in the event of prolonged replication stress. While modulation of DSB repair pathways represents one of the resistance mechanisms to chemotherapeutic drugs, maintenance of fork stability is critical to prevent carcinogenesis from developing in the first place. Here, we have identified novel post translational modifications of RIF1 that are critical for its role in the maintenance of genome stability. Specifically, phosphorylation of a conserved cluster of SQ sites in RIF1 modulates its role in fork stabilization while being dispensable for its function in DSB repair

Expression of Connective Tissue Growth Factor after Glaucoma Filtration Surgery in a Rabbit Model

PURPOSE. Connective tissue growth factor (CTGF) appears to play a significant role in mediating fibrosis in several tissues. To gain further understanding of the role of CTGF in the scar formation that occurs after glaucoma filtering surgery (GFS), experiments were performed in a rabbit model. METHODS. Three experiments were performed: (1) CTGF and transforming growth factor (TGF)-␤ expression were measured quantitatively after GFS, using ELISA. (2) After GFS conjunctival bleb tissues were immunostained for the presence of CTGF and TGF-␤. (3) Exogenous CTGF was injected into mitomycin-C (MMC)-treated filtering blebs and the scaring response compared to TGF-␤ and physiological saline-injected blebs. RESULTS. CTGF and TGF-␤ were expressed maximally by day 5 after surgery and were both shown to be present in the bleb tissues after GFS. The addition of exogenous CTGF and TGF-␤ increased the rate of failure of GFS blebs. CONCLUSIONS. These data support the hypothesis that CTGF plays an important role in scarring and wound contracture after GFS. Inhibition of CTGF synthesis or its action may help prevent bleb failure and improve long-term GFS outcomes

Inflammation-induced PELP1 expression promotes tumorigenesis by activating GM-CSF paracrine secretion in the tumor microenvironment

The inflammatory tumor microenvironment has been implicated as a major player fueling tumor progression and an enabling characteristic of cancer, proline, glutamic acid, and leucine-rich protein 1 (PELP1) is a novel nuclear receptor coregulator that signals across diverse signaling networks, and its expression is altered in several cancers. However, investigations to find the role of PELP1 in inflammation-driven oncogenesis are limited. Molecular studies here, utilizing macrophage cell lines and animal models upon stimulation with lipopolysaccharide (LPS) or necrotic cells, showed that PELP1 is an inflammation-inducible gene. Studies on the PELP1 promoter and its mutant identified potential binding of c-Rel, an NF-κB transcription factor subunit, to PELP1 promoter upon LPS stimulation in macrophages. Recruitment of c-Rel onto the PELP1 promoter was validated by chromatin immunoprecipitation, further confirming LPS mediated PELP1 expression through c-Rel–specific transcriptional regulation. Macrophages that overexpress PELP1 induces granulocyte–macrophage colony-stimulating factor secretion, which mediates cancer progression in a paracrine manner. Results from preclinical studies with normal–inflammatory–tumor progression models demonstrated a progressive increase in the PELP1 expression, supporting this link between inflammation and cancer. In addition, animal studies demonstrated the connection of PELP1 in inflammation-directed cancer progression. Taken together, our findings provide the first report on c-Rel–specific transcriptional regulation of PELP1 in inflammation and possible granulocyte–macrophage colony-stimulating factor–mediated transformation potential of activated macrophages on epithelial cells in the inflammatory tumor microenvironment, reiterating the link between PELP1 and inflammation-induced oncogenesis. Understanding the regulatory mechanisms of PELP1 may help in designing better therapeutics to cure various inflammation-associated malignancies