Interrelación entre la estructura de la cromatina, la transcripción génica y la iniciación de la replicación del DNA en células de mamífero y en Leishmania major

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-03-2017La replicación del DNA consiste en la duplicación de la información genética codificada en el genoma de las células para poder ser transmitida a su descendencia. Este proceso debe producirse una única vez a lo largo del ciclo celular y se inicia en los orígenes de replicación. El reconocimiento de estos sitios de iniciación se realiza mediante complejos proteicos denominados replicadores cuyo mecanismo de acción esta conservado desde levaduras, como Saccharomyces cerevisiae, donde el replicador reconoce una secuencia consenso, a células de metazoos en los que la especificidad de unión y actividad de los replicadores parece depender de una combinación de múltiples factores. El comienzo de la replicación en estos sistemas está estrechamente relacionado con otros procesos nucleares, especialmente con la transcripción génica, así como con la composición y estructura de la cromatina de las regiones en que se sitúan los orígenes de replicación. Para profundizar en las relaciones entre estos procesos, hemos combinado una aproximación a escala genómica de los datos disponibles en la literatura correspondientes a marcas epigenéticas, motivos y estructuras secundarias de la secuencia de DNA en los orígenes de replicación más eficientes de sistemas celulares de mamíferos con estudios detallados a alta resolución de la estructura de la cromatina y la abundancia de intermediarios de replicación en orígenes específicos. De esta manera, hemos podido comprobar cómo estos orígenes eficientes se encuentran enriquecidos en variantes de histonas que forman nucleosomas lábiles así como en motivos de secuencia característicos de cromatina accesible que, en conjunto, facilitan el reclutamiento de los complejos de reconocimiento del origen así como del resto de factores que desencadenan la síntesis del DNA. También hemos realizado análisis genómicos del perfil nucleosomal y de la abundancia de intermediarios de replicación de células promastigotes del parásito Leishmania major. La integración de estos resultados con datos del transcriptoma y de la localización de marcas epigenéticas disponibles en la literatura, nos ha permitido concluir que la transcripción activa es el determinante principal que subyace a la organización espacial y temporal de la replicación del genoma de este parásito. Estos resultados evidencian la naturaleza oportunista del proceso replicativo y sugieren que el acoplar el inicio de la replicación a la elongación de la transcripción podría ser una solución ancestral utilizada por las células eucariotas para replicar su material genético.DNA replication consists in the duplication of the genetic information encoded within the cell genome for being transmitted to the next generation. This process must to occur once during the cell cycle and starts at the replication origins. Recognition of these initiation sites is performed by protein complexes called replicators, sharing similar mechanism from yeast -like Saccharomyces cerevisiae where the replicator recognizes one consensus sequence- to metazoan cells -where the binding specificity seems to depend on a combination of several factors. Replication initiation in these systems is highly related to other nuclear processes, specially transcription, and with the chromatin structure at those regions were replication origins are situated. To go deeper into the relationship among these features, we have combined a genome-wide approach using published data for epigenetic marks, motifs and secondary structures of the DNA in the most efficient mammalian replication origins with high-resolution studies about the chromatin structure and the replication intermediates abundance at specific origins. In such a way, we have shown how these efficient origins are enriched in histone variants associated to labile nucleosomes and in sequence motifs characteristic for accessible chromatin. Both together supply the conditions for the recruitment of the origin recognition complexes and the rest of the protein factors that fire the DNA synthesis. In addition, we have already performed genome-wide analysis of the nucleosomal profile and of the replication intermediates abundance using promastigotes from the human parasite Leishmania major. Integrating these results with the transcriptome and the location of some epigenetic marks leads to the conclusion that active transcription is the main determinant underlying the spacio-temporal organization of the replication in this system. These results evince the opportunistic nature under the replication of the DNA and suggest that the coupling of it to the transcription elongation may be an ancestral way to perform the replication of the entire genetic material

Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expressionThis work was supported by grants (to B.A. and J.M.R.) from Ministerio de Economía, Industria y Competitividad, project number SAF2017-86965-R (co-funded with FEDER funds), and by the Network of Tropical Diseases Research RICET (RD16/0027/0008), co-funded with FEDER funds. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santande

Strand‐specific, high‐resolution mapping of modified RNA polymerase II

Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3′, with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3′ ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes

R-loops and initiation of DNA replication in human cells: a missing link?

The unanticipated widespread occurrence of stable hybrid DNA/RNA structures (R-loops) in human cells and the increasing evidence of their involvement in several human malignancies have invigorated the research on R-loop biology in recent years. Here we propose that physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. Quite likely, this occurs by the strand-displacement reaction activating the formation of G-quadruplex structures that target the origin recognition complex (ORC) in the single-stranded conformation. In agreement with this, we found that R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins, precisely at the position displaying the highest density of G4 motifs. This scenario builds on the connection between transcription and replication in human cells and suggests that R-loop dysregulation at CpG island promoter-origins might contribute to the phenotype of DNA replication abnormalities and loss of genome integrity detected in cancer cells.Work in our laboratory is supported by the Spanish Ministry of Economy and Competitiveness (BFU2013-45276). RL and RA are funded by grants from the Spanish Ministry of Economy and Competitiveness (BFU2010-18992) and the Portuguese Foundation for Science and Technology (SFRH/BD/81027/11), respectively.Peer reviewedPeer Reviewe

High-resolution analysis of DNA synthesis start sites and nucleosome architecture at efficient mammalian replication origins

DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high-resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island-associated origins always occur at positions of high-nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios. © 2013 European Molecular Biology Organization.Spanish Ministry of Economy and Competitiveness (BFU2010‐18992), (BFU2012‐40020); Portuguese Foundation for Science and Technology (SFRH/BD/81027/11).Peer Reviewe

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance