Daniel Gómez-Cabello

Regulation of the MicroRNA Processor DGCR8 by the Tumor Suppressor ING1

The ING family of tumor suppressor proteins controls several cellular functions relevant to antitumor protection, such as cell cycle control, apoptosis, senescence, or migration. ING proteins are functionally linked to the p53 pathway, and they participate in transcriptional control via the recognition of histone marks and recruitment of protein complexes with chromatin-modifying activity to specific promoters. Here, we have investigated the global effect of ING1 in gene regulation through genome-wide analysis of expression profiles in primary embryonic fibroblasts deficient for the Ing1 locus. We find that Ing1 has a predominant role as transcriptional repressor in this setting, affecting the expression of genes involved in a variety of cellular functions. Within the subset of genes showing differential expression, we have identified DGCR8, a protein involved in the early steps of microRNA biogenesis. We show that ING1 binds to the DGCR8 promoter and controls its transcription through chromatin regulation. We also find that ING1 and DGCR8 can cooperate in restraining proliferation. In summary, this study reveals a novel connection between ING1 and a regulator of microRNA biogenesis and identifies new links between tumor suppressor proteins and the microRNA machinery.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

DGCR8‐mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts

The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell‐cycle inhibitor p21CIP1. We further show that a subset of senescence‐associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8‐mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell‐cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.

ING proteins in cellular senescence.

Cellular senescence is an effective anti-tumor barrier that acts by restraining the uncontrolled proliferation of cells carrying potentially oncogenic alterations. ING proteins are putative tumor suppressor proteins functionally linked to the p53 pathway and to chromatin regulation. ING proteins exert their tumor-protective action through different types of responses. Here, we review the evidence on the participation of ING proteins, mainly ING1 and ING2, in the implementation of the senescent response. The currently available data support an important role of ING proteins as regulators of senescence, in connection with the p53 pathway and chromatin organization.

A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection

There are two major and alternative pathways to repair DNA double-strand breaks: non-homologous end-joining and homologous recombination. Here we identify and characterize novel factors involved in choosing between these pathways; in this study we took advantage of the SeeSaw Reporter, in which the repair of double-strand breaks by homology-independent or -dependent mechanisms is distinguished by the accumulation of green or red fluorescence, respectively. Using a genome-wide human esiRNA (endoribonuclease-prepared siRNA) library, we isolate genes that control the recombination/end-joining ratio. Here we report that two distinct sets of genes are involved in the control of the balance between NHEJ and HR: those that are required to facilitate recombination and those that favour NHEJ. This last category includes CCAR2/DBC1, which we show inhibits recombination by limiting the initiation and the extent of DNA end resection, thereby acting as an antagonist of CtIP.

CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells

Summary Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs). All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.

RAD51 paralogs regulate double strand break repair pathway choice by limiting Ku complex retention

RAD51 paralogs are a group of conserved proteins in eukaryotes that are involved in the repair of DNA breaks at several levels. On one hand, they help the strand invasion step catalyzed by RAD51. Also, they play late roles in Holliday Junction metabolism. Here we uncover a new role of the RAD51 paralogs at an earlier event in the repair of broken chromosomes. All five RAD51 paralogs affect the balance between double strand break repair pathways. Specifically, they favor homology-mediated repair over non-homologous end-joining. Such role is independent of RAD51 or the checkpoint activity of these proteins. Moreover, it defines a novel control point of double strand break repair independent and subsequent to DNA-end resection initiation. Mechanistically, RAD51 paralogs limit the retention of Ku80 at the sites of DNA breaks. Thus, our data extend the role of this family of proteins to the earliest event of double strand break repair.