Background: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21^WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. Results: We now demonstrate that p21^WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. Conclusions: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21^WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

Background: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrentin situanalysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have renderedin situco-detection unfeasible until now.Methods: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGor^TMcompound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs.Results: This experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all correspondingin vitromodels are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics.Conclusions: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to Gfp and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real-time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real-time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.