Nucleosome Positioning in Budding Yeast = Posicionamiento de nucleosomas en Saccharomyces cerevisiae

Abstract

[spa] Nuestro estudio se centra en el posicionamiento de nucleosomas a nivel genómico en levadura, con tal de explorar los factores determinantes de nucleosomas y su plasticidad a lo largo del ciclo celular, así como su relación con la expresión génica basándonos en la cantidad de mARN celular. Encontramos que las regiones libres de nucleosomas (NFRs en inglés) en 5’ y 3’ contienen propiedades físicas inusuales, las cuales son intrínsecas del ADN genómico. Además, demostramos que estas propiedades físicas actúan sinérgicamente con factores de transcripción para definir las NFRs. Una vez la NFR está definida, el posicionamiento de nucleosomas en torno al inicio de transcripción (TSS en inglés) puede predecirse con modelos estadísticos simples. No obstante, también observamos que los nucleosomas son bastante dinámicos en las regiones distales a 5’NFRs y poseen distintos mecanismos reguladores. Nuestro análisis comparativo acerca de la organización de los nucleosomas reveló que la cromatina de hecho exhibe una configuración distinta debido al reordenamiento dependiente de la replicación en fase S, mostrando una mayor sensibilidad de corte por el enzima MNase y un mayor número de nucleosomas deslocalizados a lo largo del genoma. Adicionalmente, observamos características particulares en fase M, donde la cromatina sufre un mayor grado de compactación. Notablemente, estos cambios en la organización de la cromatina son repentinos y agudos y sólo afectan a algunas regiones del genoma, mientras que la mayoría de genes presentan una conservación del patrón de nucleosomas a lo largo del ciclo celular. El análisis detallado en torno a los orígenes de replicación muestra una NFR más ancha en fase G1, debido a la unión del complejo pre-replicatorio. Una vez se activa el origen, los nucleosomas sólo ocupan parcialmente la NFR, debido a la unión constitutiva del complejo de origen de replicación (ORC en inglés). También proporcionamos evidencias de que los orígenes tempranos tienden a tener una organización nucleosomal más ordenada que los tardíos. Finalmente, ilustramos que los nucleosomas centroméricos poseen un posicionamiento idóneo y asimismo, un ensamblaje distinto. Sin embargo, nuestro análisis también mostró la dinámica de los nucleosomas centroméricos a lo largo del ciclo celular, indicando que de hecho su composición puede oscilar a lo largo del ciclo celular. En conjunto, nuestro detallado estudio proporciona una imagen dinámica del posicionamiento de nucleosomas y sus factores determinantes; nuevos indicios respecto a la organización de la cromatina en regiones reguladoras clave en base al ciclo celular y su conexión con la expresión génica; y finalmente, añade una nueva dimensión a la caracterización de los nucleosomas centroméricos.

[eng] The nucleosome is the fundamental structural unit of DNA compaction in eukaryotic cells and is formed by the wrapping of 147 bp double stranded DNA around a histone octamer. Nucleosome organization plays a major role in controlling DNA accessibility to regulatory proteins, hence affecting cellular processes such as transcription, DNA replication and repair. Our study focuses on genome-wide nucleosome positioning in S. cerevisiae to explore nucleosome determinants and plasticity throughout the cell cycle and their interplay with gene expression based on cell mRNA abundance. We pursued the contribution of DNA physical properties on nucleosome organization around key regulatory regions such as TSSs and TTSs by analyzing genome-wide MNase-digestion profile of genomic DNA. We also implemented a systematic approach to standardize MNase-Seq experiments by minimizing the noise generated by extrinsic factors to enable an accurate analysis of the underlying principles of nucleosome positioning and dynamics. Moreover, we carried out a large-scale study of nucleosome plasticity throughout the cell cycle and its interplay with transcription based on a comparative analysis among nucleosome maps, gene expression data and MNase sensitivity assays. We then focused on nucleosome organization around DNA replication origins and its possible effect on origin activation. Finally, we sought to characterize centromeric nucleosome composition and its oscillation along cell cycle. During the course of these studies, we found that key regulatory regions such as 5’ and 3’ nucleosome free regions (NFRs) contain unusual physical properties that are intrinsic to genomic DNA. We further demonstrated that DNA physical properties and transcription factors act synergistically to define NFRs, especially in genes with an open promoter structure. Once NFR is defined, the nucleosome positioning around TSSs can be predicted by a simple statistical model, supporting the energy barrier model for nucleosome positioning determination. However, we also observed that nucleosomes are quite dynamic at distal 5’ NFRs and do have distinct regulatory mechanisms. Our comparative analysis of nucleosome organization along cell cycle revealed that chromatin exhibits a distinct configuration due to DNA replication-dependent organization at S phase, showing higher sensitivity to MNase and displaying fuzzier nucleosomes along the genome. Moreover, we observed different features at M phase, where chromatin compaction is the highest and displays a slightly different pattern than in G1 and G2 phases. Interestingly, these changes in chromatin organization are sudden and acute and only affect some regions of the genome, whereas the majority of genes present conserved nucleosome patterns along cell cycle. Our individual gene analysis disclosed that the largest changes take place in cell cycle-dependent genes, indicating the interplay between chromatin and transcription. Moreover, a distinct nucleosome organization at high and low transcription rates further supports this relationship. The detailed analysis around replication origins shows that they display slightly wider NFRs at G1 phase due to pre-Replication complex binding. Once the replication origins are active, nucleosomes partially occupy NFRs up to a certain extent due to constitutive binding of ORC. Moreover, we provided further evidence that early firing origins tend to have more ordered nucleosome organization than late firing origins. Finally we illustrated that centromeric nucleosomes display a perfect positioning, confirming their strong centromeric sequence-dependent recruitment to DNA. The characterization of histone composition under physiological cell conditions suggested that the octameric nucleosome assembly model is favored in centromeres. Yet, our analysis along cell cycle showed centromeric nucleosome dynamics, proposing that its composition might oscillate along cell cycle. Taken together, our accurate study provides a dynamic picture of nucleosome positioning and its determinants; new insights into cell cycle-dependent chromatin organization on key regulatory regions and its interplay with gene expression; and adds a new dimension to the characterization of centromeric nucleosomes.