RanBP2-Mediated SUMOylation Promotes Human DNA Polymerase Lambda Nuclear Localization and DNA Repair

Cellular DNA is under constant attack by a wide variety of agents, both endogenous and exogenous. To counteract DNA damage, human cells have a large collection of DNA repair factors. Among them, DNA polymerase lambda (Polλ) stands out for its versatility, as it participates in different DNA repair and damage tolerance pathways in which gap-filling DNA synthesis is required. In this work we show that human Polλ is conjugated with Small Ubiquitin-like MOdifier (SUMO) proteins both in vitro and in vivo, with Lys27 being the main target of this covalent modification. Polλ SUMOylation takes place in the nuclear pore complex and is mediated by the E3 ligase RanBP2. This post-translational modification promotes Polλ entry into the nucleus, which is required for its recruitment to DNA lesions and stimulated by DNA damage induction. Our work represents an advance in the knowledge of molecular pathways that regulate cellular localization of human Polλ, which are essential to be able to perform its functions during repair of nuclear DNA, and that might constitute an important point for the modulation of its activity in human cells

Regulation of human Polλ by ATM-mediated phosphorylation during Non-Homologous End Joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.España MINECO y la Comisión Europea (European Regional Development Fund) to J.F.R. RYC-2011-08752, BFU2013-44343-P) and to F.C-L. (SAF2014-55532-R)

Identificación de genes asociados a Epilepsia mediante secuenciación de exoma

Trabajo Fin de Grado.Peer Reviewe

Uso del Killifish como modelo de investigación en la biomedicina

Comunicación presentada en la XXVI CONVENCIÓN Sociedad Española de Killifish, celebrada en El Cuervo de Sevilla (España), del 26 al 28 de abril de 2019

Partial functional divergence of the paralog genes znf503 and znf703 during zebrafish embryonic development

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en Sevilla (España) del 18 al 21 de septiembre de 2023.Peer reviewe

Role of transcription factors in gene expression regulation during zebrafish embryonic development

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022

Impact of signaling pathways in the cis-regulation during embryonic development

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022.The animal body plan formation takes place during the embryonic development and it results from the precise spatio-temporal regulation of the gene expression. The animal form evolves largely by altering the expression of functionally conserved developmental genes and the vast networks they control. Most of the genomic signatures underlying key evolutionary processes can be found in the cis-regulatory elements (CREs) that control when, where and how much the genes are transcribed. We have identified genome-wide CREs acting during early development in zebrafish at different embryonic stages. To do that, we performed both ATAC-seq and RNA-seq, which allowed us to link the identified CREs to their target developmental genes and determine how their activity change in the different developmental stages. Several works over the last decades have shown that regulatory DNA can act at long distances, often more than 1 Mb, allowing the contact with promoters of target genes through chromatin loops. These enhancer-promoter physical interactions occur in a highly regulated manner, showing the importance of a precise regulation of the gene expression along development and animal evolution. To identify activated or repressed enhancers upon modulation of the genetic pathways, embryos have been treated with different drugs during the gastrulation period, when body plan basic layout is generated. At this point the open chromatin profiling have been done at three different stages: 80% of epiboly (gastrulation), 12 somites (neurulation) and 24hpf stage (organogenesis). Altogether, the goal of this project is to understand how developmental signaling pathways control CREs activity and regulate gene expression over different stages during the embryonic development

Multianalytical characterization of Late Roman glasses including nanosecond and femtosecond laser induced breakdown spectroscopy

In the present study, a historical set of Late Roman glasses from a recently unearthed graveyard located in the small city of Cubas de la Sagra, within the Madrid region (Spain) was compositionally analysed using different techniques such as ultraviolet-visible (UV-Vis) and laser induced fluorescence (LIF) spectroscopy, X-ray fluorescence (XRF) and laser induced breakdown spectroscopy (LIBS). LIBS results, recorded upon nanosecond (ns) and femtosecond (fs) laser irradiation, served for identification of major glass components (to classify them into main historical glass groups) and of minor components (e.g. chromophores, decolouring agents and degradation products). Quantitative information regarding these components was obtained on the basis of calibration curves obtained using glass certified standards and local standards. We have demonstrated that LIBS serves for the non-invasive/micro-destructive, quantitative chemical characterization of most of the analysed historical glasses. Furthermore, this work establishes a comparison between LIBS analysis of glasses in the ns and fs regimes on one hand, and on the other hand with the results obtained using XRF. The procedures and protocols here proposed can be applied for in situ study of historical glass collections, regardless of their size, provenance and chronology.This work was funded by MINECO (Project CTQ2013-43086-P, HAR2012-30769 and FIS2013- 44174-P) and Programa Geomateriales 2-CM (CAM, S2013/MIT-2914) and counted with the support from Techno-Heritage (Spanish Network of Science and Technology for Heritage Conservation).Peer reviewe

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Commission (European Regional Development Fund) to J.F.R. (RYC-2011-08752, BFU2013-44343-P) and to F.C-L. (SAF2014-55532-R). J.F.R. was the recipient of a Ramón y Cajal contract from the Spanish Ministry of Economy and Competitiveness (MINECO). G.S.-M. was supported by a JAE-predoctoral fellowship from the Spanish Research Council (CSIC). Work by J.M.P in the Ramsden laboratory was supported by a postdoctoral fellowship from the American Cancer Society (PF-14-0438-01-DMC) and an NCI grant (CA097096) to D.A.R.Peer Reviewe

Deciphering the gene regulatory network of retinoic acid signaling during zebrafish embryonic development.

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021.Retinoic acid (RA) is the active form of retinol (vitamin A) and functions as a ligand for the nuclear RA receptors (RARs), which regulate the expression of target genes by binding to RA response elements (RAREs). RA signaling is required for embryonic development in chordate animals, being involved in multiple processes, such as body axis extension, hindbrain antero-posterior (AP) patterning, differentiation of neuro-mesodermal progenitors, heart AP patterning and forelimb bud initiation, among others. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during embryonic development. Here we use ChIP-seq to profile the genomic binding of RAR in zebrafish embryos at different developmental stages. Stimulating the RA pathway by treating embryos with all-trans-RA (atRA), the most abundant form of RA, we use a combination of RNA-seq, ATAC-seq and ChIP-seq to gain insight into the mechanisms by which RA signaling control the expression of direct and indirect target genes during early development. Finally, we explore the interplay between RA signaling, Hox and Meis transcription factors, which have been proposed to cooperate during axial skeleton AP patterning. Our results provide new clues about the crosstalk between signaling pathways and transcription factors during vertebrate embryonic development