Computational analysis of transcriptional regulation in metazoans

This HDR thesis presents my work on transcriptional regulation in metazoans (animals). As a computational biologist, my research activities cover both the development of new bioinformatics tools, and contributions to a better understanding of biological questions. The first part focuses on transcription factors, with a study of the evolution of Hox and ParaHox gene families across meta- zoans, for which I developed HoxPred, a bioinformatics tool to automatically classify these genes into their groups of homology. Transcription factors regulate their target genes by binding to short cis-regulatory elements in DNA. The second part of this thesis introduces the prediction of these cis-regulatory elements in genomic sequences, and my contributions to the development of user- friendly computational tools (RSAT software suite and TRAP). The third part covers the detection of these cis-regulatory elements using high-throughput sequencing experiments such as ChIP-seq or ChIP-exo. The bioinformatics developments include reusable pipelines to process these datasets, and novel motif analysis tools adapted to these large datasets (RSAT peak-motifs and ExoProfiler). As all these approaches are generic, I naturally apply them to diverse biological questions, in close collaboration with experimental groups. In particular, this third part presents the studies uncover- ing new DNA sequences that are driving or preventing the binding of the glucocorticoid receptor. Finally, my research perspectives are introduced, especially regarding further developments within the RSAT suite enabling cross-species conservation analyses, and new collaborations with exper- imental teams, notably to tackle the epigenomic remodelling during osteoporosis.Cette thèse d’HDR présente mes travaux concernant la régulation transcriptionelle chez les métazoaires (animaux). En tant que biologiste computationelle, mes activités de recherche portent sur le développement de nouveaux outils bioinformatiques, et contribuent à une meilleure compréhension de questions biologiques. La première partie concerne les facteurs de transcriptions, avec une étude de l’évolution des familles de gènes Hox et ParaHox chez les métazoaires. Pour cela, j’ai développé HoxPred, un outil bioinformatique qui classe automatiquement ces gènes dans leur groupe d’homologie. Les facteurs de transcription régulent leurs gènes cibles en se fixant à l’ADN sur des petites régions cis-régulatrices. La seconde partie de cette thèse introduit la prédiction de ces éléments cis-régulateurs au sein de séquences génomiques, et présente mes contributions au développement d’outils accessibles aux non-spécialistes (la suite RSAT et TRAP). La troisième partie couvre la détection de ces éléments cis-régulateurs grâce aux expériences basées sur le séquençage à haut débit comme le ChIP-seq ou le ChIP-exo. Les développements bioinformatiques incluent des pipelines réutilisables pour analyser ces jeux de données, ainsi que de nouveaux outils d’analyse de motifs adaptés à ces grands jeux de données (RSAT peak-motifs et ExoProfiler). Comme ces approches sont génériques, je les applique naturellement à des questions biologiques diverses, en étroite collaboration avec des groupes expérimentaux. En particulier, cette troisième partie présente les études qui ont permis de mettre en évidence de nouvelles séquences d’ADN qui favorisent ou empêchent la fixation du récepteur aux glucocorticoides. Enfin, mes perspectives de recherche sont présentées, plus particulièrement concernant les nouveaux développements au sein de la suite RSAT pour permettre des analyses basées sur la conservation inter-espèces, mais aussi de nouvelles collaborations avec des équipes expérimentales, notamment pour éudier le remodelage épigénomique au cours de l’ostéoporose

Reassessment of Piwi Binding to the Genome and Piwi Impact on RNA Polymerase II Distribution

Drosophila Piwi was reported by Huang et al. (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits Heterochromatin Protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang et al. (2013) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov et al. (2015) disputed the validity of the Huang et al. bioinformatic pipeline that led to the last two claims. Here we report our independent reanalysis of the data using current bioinformatic methods. Our reanalysis agrees with Marinov et al. (2015) that Piwi’s genomic targets still remain to be identified, yet confirms the Huang et al. claim that Piwi influences RNA polymerase II distribution in the genome. This Response addresses the Marinov et al. (2015) Matters Arising, published concurrently in Developmental Cell

The evolutionary genomics of CTCF binding and functional signatures in mouse.

Genetic differences within and between species predominantly lie in the noncoding sequence of the regulatory regions of the genome whose function and significance largely remain poorly understood. Despite significant progress in the field of genomics and the rapid progress in sequencing methods and the subsequent explosion of genomic data, our understanding of the role of the non- coding genetic sequence in the regulation of tissue- and species-specific gene expression is still lagging behind, limiting our comprehension of the evolutionary mechanisms and pressures that shape those expression profiles, and their involvement in the health and disease. The CTCF protein demarcates mammalian genomes into discrete transcriptionally active domains, providing the platform for complex spatial and temporal regulatory processing of genetic information that govern biological processes. In this thesis, I investigate the dynamics and functional implications of evolutionarily novel CTCF binding sites in two Mus genus mouse subspecies, Mus musculus domesticus and Mus musculus castaneus, separated by a short evolutionary time of only one million years. The project investigated the subspecies-specific binding of CTCF in terms of the repeat content, evolution, functional impact and involvement in chromatin conformation. The key findings of this investigation are: (1) the incorporation of young CTCF sites into the non-coding genome via action of transposable elements is followed rapidly with the exhibition of various characteristics of biological function; (2) Unlike other tissue-specific transcription factors, allele- specific CTCF occupancy is affected by cis- and trans-acting regulatory mechanisms that exhibit similar functional characteristics; (3) CTCF evolutionary dynamics support both maintenance of pre-existing structures and functions and provide template for novel ones. In summary, this thesis discusses the evolutionary dynamics of CTCF genomic occupancy and functional signatures in short evolutionary time, and illustrates how either novel species-specific CTCF sites, or common sites with newly-acquired genotypic variants integrate into existing genomic architecture and begin to exert their effects