Nucleosome positioning on chromatin using bioinformatics techniques = Posicionament de nucleosomes en cromatina emprant tècniques bioinformàtiques

Abstract

This thesis presents a broad study of the chromatin structure and function with special focus in the nucleosome organization. More specifically, it includes three different but complementary perspectives: i) Analysis of the DNA from a physical point of view, correlating chromatin structure with DNA function and its intrinsic properties, ii) Development of tools to extract information on nucleosome position and dynamics from experimental data —mostly Next Generation Sequencing experiments— and iii) In depth analysis of chromatin structure and dynamics in model systems (such as S.Cerevisae, C.Elegans or, in a smaller extent, Human).

In the first work, “Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast”, we show how a simple energetic predictor accounting for primary sequence and a mesoscopic model of DNA mechanics can describe the nucleosome positioning in key regulatory areas. Furthermore, we describe how these principles also affect the endo/exo nuclease activity of Microccocal Nuclease and their sequence affinity without substantially biasing the nucleosome maps. In the second work, “Impact of Methylation on the Physical Properties of DNA”, we show how DNA methylation can alter the mechanistic properties and therefore change the affinity of different sequences for nucleosomes, providing in vitro evidences of this relationship. The same descriptors were used before to predict promoters in human with a good sensitivity but minor sensibility. In the third work, “Unraveling the hidden DNA structural/physical code provides novel insights on promoter location“, we present how the false positives obtained in the previous work are indeed transcriptionally active regions of the genome without any annotated gene previously.

In the second block we studied the nucleosome organization in vivo in different conditions. In the work “Nucleosome architecture and plasticity along cell cycle”, we provide new insights about the nucleosome dynamics during the process of DNA replication and chromosome segregation. In general, our results show an increased disorganization of chromatin during the replication stage and a gene-specific interplay of nucleosomes and gene expression levels. In the work “Fuzziness and noise in nucleosomal architecture” we use different nucleosomes maps obtained during the cell cycle to evaluate the general reproducibility and quantification of nucleosome phasing. Results show how synchronized populations of cells are less noisy, pointing also to technical factors, which can increase the fuzziness of nucleosomes maps. Furthermore, we provide a new insight on how physical properties of DNA affect different nucleosomes architectures and this interplays with in vivo factors to determine the nucleosome positioning. Additionally, two collaborations with external groups are presented, “Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling” and “Modeling genome-wide kinetics of endo/exonuclease digestion”.

In the methodological part, we present four new R/Bioconductor packages for the study of chromatin and integrative analysis. The first, “nucleR: a package for non-parametric nucleosome positioning” is a fast and efficient nucleosome caller for tiling arrays and next generation sequencing experiments. “htSeqTools: high-throughput sequencing quality control, processing and visualization in R” is a library for the quality control and common tasks related with next generation sequencing experiments. “DASiR: Programmatic data retrieval from DAS servers in R” is a R convenient interface for servers implementing the Distrubuted Annotation System. Finally, “Dynamic analysis of nucleosome positioning at read level”, presents a new tool for the read-level analysis of nucleosome positions allowing the inference and annotation of the dynamics between two reference maps.