Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcriptionreplication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase d, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution. the paper; M.L. and A.P. revised and modified the paper.

Type I interferon (IFN) is essential for host defenses against viruses; however, dysregulated IFN signaling is causally linked to autoimmunity, particularly systemic lupus erythematosus. Autoimmune disease treatments rely on glucocorticoids (GCs), which act via the GC receptor (GR) to repress proinflammatory cytokine gene transcription. Conversely, cytokine signaling through cognate Jak/STAT pathways is reportedly unaffected or even stimulated by GR. Unexpectedly, we found that GR dramatically inhibited IFN-stimulated gene (ISG) expression in macrophages. The target of inhibition, the heterotrimeric STAT1-STAT2-IRF9 (ISGF3) transcription complex, utilized the GR cofactor GRIP1/TIF2 as a coactivator. Consequently, GRIP1 knockdown, genetic ablation, or depletion by GC-activated GR attenuated ISGF3 promoter occupancy, preinitiation complex assembly, and ISG expression. Furthermore, this regulatory loop was restricted to cell types such as macrophages expressing the GRIP1 protein at extremely low levels, and pharmacological disruption of the GR-GRIP1 interaction or transient introduction of GRIP1 restored RNA polymerase recruitment to target ISGs and the subsequent IFN response. Thus, type I IFN is a cytokine uniquely controlled by GR at the levels of not only production but also signaling through antagonism with the ISGF3 effector function, revealing a novel facet of the immunosuppressive properties of GCs.

Inhibition of cytokine gene expression by the hormone-activated glucocorticoid receptor (GR) is the key component of the antiinflammatory actions of glucocorticoids, yet the underlying molecular mechanisms remain obscure. Here we report that glucocorticoid repression of cytokine genes in primary macrophages is mediated by GR-interacting protein (GRIP)1, a transcriptional coregulator of the p160 family, which is recruited to the p65-occupied genomic NFκB-binding sites in conjunction with liganded GR. We created a mouse strain enabling a conditional hematopoietic cell-restricted deletion of GRIP1 in adult animals. In this model, GRIP1 depletion in macrophages attenuated in a dose-dependent manner repression of NFκB target genes by GR irrespective of the upstream Toll-like receptor pathway responsible for their activation. Furthermore, genome-wide transcriptome analysis revealed a broad derepression of lipopolysaccharide (LPS)-induced glucocorticoid-sensitive targets in GRIP1-depleted macrophages without affecting their activation by LPS. Consistently, conditional GRIP1-deficient mice were sensitized, relative to the wild type, to a systemic inflammatory challenge developing characteristic signs of LPS-induced shock. Thus, by serving as a GR corepressor, GRIP1 facilitates the anti-inflammatory effects of glucocorticoids in vivo.inflammation | macrophage transcriptome | transcriptional regulation | coactivators | corepressors

Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2 0 -deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon c (IFNc), IFNb and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/ chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNb-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.