Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.This work was supported by Grants MPY-1038/14 to Alberto Zambrano, PI 11/00590 to Isidoro Martínez and RD12/0036/0030 to Ana Aranda from FIS (Instituto de Salud Carlos III) and Grant BFU2011-28958, from Ministerio de Economía y Competitividad to Ana Aranda. The authors have no conflicting financial interests

CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation

Abstract Background The reported antitumor activity of the BET family bromodomain inhibitors has prompted the development of inhibitors against other bromodomains. However, the human genome encodes more than 60 different bromodomains and most of them remain unexplored. Results We report that the bromodomains of the histone acetyltransferases CREBBP/EP300 are critical to sustain the proliferation of human leukemia and lymphoma cell lines. EP300 is very abundant at super-enhancers in K562 and is coincident with sites of GATA1 and MYC occupancy. In accordance, CREBBP/EP300 bromodomain inhibitors interfere with GATA1- and MYC-driven transcription, causing the accumulation of cells in the G0/G1 phase of the cell cycle. The CREBBP/CBP30 bromodomain inhibitor CBP30 displaces CREBBP and EP300 from GATA1 and MYC binding sites at enhancers, resulting in a decrease in the levels of histone acetylation at these regulatory regions and consequently reduced gene expression of critical genes controlled by these transcription factors. Conclusions Our data shows that inhibition of CREBBP/EP300 bromodomains can interfere with oncogene-driven transcriptional programs in cancer cells and consequently hold therapeutic potential

MOESM1 of CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation

Additional file 1: Table S1. gRNAs used for gene editing. Fig. S1. Statistical analysis of the CRISPR-Cas9 growth competition experiments. Tukey Kramer analysis of the adjusted percentages of growth inhibition caused by gRNAs targeting different regions of CREBBP (A) or EP300 (B). 5′ coding region (5′), non-conserved aminoacids of the bromodomain (ncBD), conserved aminoacids of the bromodomain (cBD) and non-target (NT). Fig. S2. Enrichment of gene expression changes after treatment with CBP30 and I-CBP112. (A) p-values for enrichment of SE-associated genes (SE) and genes with top levels of EP300 (EP300) in genes upregulated and downregulated by CBP30 and I-CBP112 treatments. (B) GSEA analysis of changes in gene expression caused by the indicated treatments and gene sets. Fig. S3. GATA1 mRNA expression in cancer cell lines and patients. (A) mRNA levels of GATA1 determined by microarray in CCLE lines grouped by cancer type. (B) GATA1 mRNA levels determined by RNAseq in cancer patients according to TCGA. Fig. S4. Expression of GATA1 splicing variants in K562 (A) Three variants are expressed in K562 according to the analysis of the RNA-seq experiment (B) Graph shows the levels of expression of the different variants in K562 cells treated with vehicle or two concentrations of CBP30. P-values for significant changes (p ≤ 0.05) are shown. Fig. S5. Human myeloma cell lines with MYC amplifications or translocations are sensitive to CBP30. (A) IC50s of growth inhibition in KMS11 or MM1S cells treated with JQ1, C646 and CBP30 for 7 days. (B) mRNA (upper panel) and protein (lower panel) levels of MYC in KMS11 or MM1S cells treated with 2 µM CBP30, 10 µM C646 and 150 nM JQ1 for 48 hours