Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells

Abstract BACKGROUND: The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. RESULTS: Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others. CONCLUSIONS: Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression

Doctor of Philosophy

dissertationCells of the early vertebrate embryo are distinct in their ability to commit into any cell lineage. How the embryo acquires this remarkable plasticity from two terminally differentiated gametes remains largely unknown. The plasticity in early embryo relies on achieving a unique transcriptome, which is regulated at multiple levels - including chromatin accessibility at developmental enhancers and genes. To understand the global landscape of chromatin accessibility during early embryogenesis, we utilized zebrafish embryos and explored three aspects of chromatin regulation. We first focused on the ATPase subunits (Brg1 and Brm) of SWI/SNF complexes, which are important regulators of chromatin accessibility and gene expression in all eukaryotes. To understand where they act in the genome, we profiled the occupancy of Brg1 and Brm by ChIP-seq at three early embryonic stages around the major onset of zygotic genome activation. We observed the occupancy of Brg1 and Brm during early embryogenesis is highly dynamic. The promoters of key pluripotency factors and other developmental transcription factors are robustly occupied by Brg1 and Brm. Interestingly, Brg1, but not Brm, is highly correlated with active histone modifications. However, only Brm commonly occupies gene bodies, which is dependent on transcription elongation. This work suggests SWI/SNF complexes might play important roles during early embryogenesis, and also reveals distinct roles of Brg1 and Brm in early zebrafish development. We then profiled the global landscape of accessible chromatin by ATAC-seq at three embryonic stages, as well as one differentiated tissue, adult liver. The data suggest chromatin accessibility increases during early embryogenesis. Here, 60% of open chromatin regions reside at genic regions and are highly enriched at promoters. Furthermore, many interesting candidate transcription factors are revealed based on motif analyses. Finally, ATAC-seq fragments with length of 120-220bp, together with MNase-seq date are used to profile nucleosome positioning. Our data determines nucleosome positioning during early embryogenesis, also discovered many interesting sequence characteristics involved in nucleosome positioning at various gene features. In summary, this work has extensively investigated the dynamics of chromatin landscape and the role of chromatin remodelers during early zebrafish development, which allow the comprehensive understanding of the regulation during early embryogenesis

On the molecular basis of mammalian totipotency

The transient capacity to autonomously form and organize all of the embryonic and extra- embryonic tissues involved in the development of a complete organism is termed totipotency. In mammals, totipotency is a feature restricted to the earliest cells of the pre-implantation embryo, which harbor this unique capacity during the first 1-3 cell cycles, depending on the species. However, our understanding of the regulatory mechanisms responsible for the establishment, maintenance and termination of such a highly plastic regulatory state remains limited. Mammalian totipotency occurs concomitantly to a set of highly-intermingled biological processes such as global chromatin remodeling, an unusual set of metabolic characteristics and the de-repression of the vast majority of transposable elements, and it is unclear whether these processes act to sustain it. Following a general overview of these processes, in this dissertation I present my contributions to a body of work on an in vitro model system for mammalian totipotency, which exhibits certain molecular features of the in vivo totipotent state. Afterwards, in the second part of this thesis, I present the transcriptional analyses that I have conducted with the aim of understanding the role of transposable element transcription during pre-implantation development. Overall, this work describes a set of phenomena that arise in totipotent cells in vivo and in totipotent-like cells in vitro and explores how recapitulating certain molecular features of totipotent cells in pluripotent cells induces a totipotent-like state in vitro

A history of why fathers' RNA matters

Having been debated for many years, the presence and role of spermatozoal RNAs is resolving, and their contribution to development is now appreciated. Data from different species continue show that sperm contain a complex suite of coding and noncoding RNAs that play a role in an individual's life course. Mature sperm RNAs provide a retrospective of spermatogenesis, with their presence and abundance reflecting sperm maturation, fertility potential, and the paternal contribution to the developmental path the offspring may follow.Sperm RNAs delivered upon fertilization provide some of the initial contacts with the oocyte, directly confront the maternal with the paternal contribution as a prelude to genome consolidation. Following syngamy, early embryo development may in part be modulated by paternal RNAs that can include epidydimal passengers. This provides a direct path to relay an experience and then initiate a paternal response to the environment to the oocyte and beyond. Their epigenetic impact is likely felt prior to embryonic genome activation when the population of sperm delivered transcripts markedly changes. Here, we review the insights gained from sperm RNAs over the years, the subtypes, and the caveats of the RNAs described. We discuss the role of sperm RNAs in fertilization and embryo development, and their possible mechanism(s) influencing offspring phenotype. Approaches to meet the future challenges as the study of sperm RNAs continues, include, elucidating the potential mechanisms underlying how paternal allostatic load, the constant adaptation of health to external conditions, may be relayed by sperm RNAs to affect future generations

Investigating the role of DNA methylation in pluripotency and differentiation using an embryoid body model

Epigenetics is the study of heritable alterations in phenotype caused by changes in cellular properties, but where the genotype is unchanged. At the molecular level these changes include chemical modifications of DNA and histones in chromatin. Specific chromatin states are associated with gene activity or silencing. The proper functioning of these mechanisms is critical for mammalian survival, particularly during embryonic development. One of the best studied epigenetic modifications is DNA methylation, wherein methyl-groups are placed on cytosines in CpG dinucleotide contexts by DNA methyltransferases to form 5mC. The malfunction of this mechanism is associated with failure of embryogenesis and many adult human disease pathologies, including cancer. However, questions remain about how the 5mC patterns are established de novo, how the patterns can change between different cell types, and why some cell types can tolerate the absence of 5mC but not others. Genetic removal of the de novo (Dnmt3a and Dnmt3b) or maintenance methyltransferases (Dnmt1) in somatic cells can lead to cell death. However, mouse embryonic stem cells (ESCs) can proliferate normally in the absence of all three of these proteins. This suggests that DNA methylation becomes essential at some point after the cells exit pluripotency and begin differentiation. Being able to identify this window of time during development is important for better understanding the dynamics of recruitment of these proteins and deposition of the mark and could therefore provide insight into how their misregulation contributes to disease. In this thesis I investigate the role of DNA methylation during ESC differentiation using a combination of conditional and reporter cell lines, in vitro differentiation models, RNA sequencing, genetic engineering, and high-resolution imaging. The ESC lines include those in which i) the expression of Dnmt1 and key pluripotency genes can be tuned separately and in combination, ii) contain germ layer differentiation reporters, and iii) contain reporters of 5mC distribution. I allow these cells to differentiate to form embryoid bodies (EBs), a validated embryogenesis-like model that enables simulation of the pluripotent-to-differentiated transition in vitro. By combining these approaches, I was able to investigate the impact of loss of Dnmt1 on gene expression pathways including apoptosis, primordial germ cell (PGC) and 2C-like cell formation, germ layer differentiation, and changes in transposable element expression. I was also able to delineate differentiation trajectories by comparing my bulk RNA sequencing data with published single cell RNA sequencing data. Overall, I observed that inhibition of Dnmt1 activity consistently led to a significant reduction in EB size though germ layer differentiation was still able to occur. Likewise, EBs were still able to form in the absence of master pluripotency factor Oct4, although to a reduced capacity. Loss of both proteins led to smaller EBs than the wild type. Interestingly Oct4-/-, Dnmt1+/+ EBs were the most affected. There were no significant changes in frequency of apoptotic cells, and only LTR-family transposable elements were de-repressed. By comparing data, I was able to identify that loss of Dnmt1 enriched EBs for PGC-like cell marker genes at late stages of differentiation. I conclude that under normal differentiation initiation conditions the two systems, the pluripotency network and the DNA methylation network, work synergistically to control the gradual switch of cells from the pluripotent to the differentiated state enabling the formation of a limited population of PGC-like cells prior to advanced differentiation of germ layer cell lineages. The purpose of this may be to protect emerging PGC-like cells from Transposon Element activity. It may also be to allow the number of pluripotent cells to reach a threshold level prior to initiating differentiation to prevent the EB size being limited in a mechanism similar to that suggested for primordial dwarfism

Molecular dissection of CTCF-associated chromatin boundaries

TAD-Grenzen sind genomische Regionen mit Isolatorpotenzial, die zwischen benachbarten Chromatindomänen liegen und deren Unterbrechung zu einer pathologischen Genexpression führen kann. Die meisten TAD-Grenzen werden durch das CTCF gebunden, ein Architekturprotein, das Chromatinschleifen bevorzugt zwischen distalen Paaren von CTCF-Bindungsstellen (CBS) mit einer konvergenten Motivausrichtung bildet. An TAD-Grenzen sind die CBS häufig geclustert, wobei die Motive eine divergente Ausrichtung aufweisen und Chromatinschleifen in Richtung der stromaufwärts und stromabwärts gelegenen Regionen projizieren. Wie die CTCF-Besetzung die Isolierung an TAD-Grenzen moduliert, ist immer noch nicht ganz klar. Hier habe ich die regulatorische Logik von CTCF-geclusterten TAD-Grenzen untersucht, indem ich genomweite Analysen und in vivo-Mausexperimente an der Epha4-Pax3-TAD-Grenze kombiniert habe. Analysen einzelner Deletionen zeigten einen deutlichen hierarchischen Beitrag von CBS zur Grenzfunktion. Im Gegensatz dazu zeigten kombinierte CBS-Deletionen ein gewisses Maß an funktioneller Redundanz und Kooperativität zwischen den Stellen. Diese Analysen zeigten auch, dass die abweichende Konfiguration der CBS, die immer wieder an TAD-Grenzen zu finden ist, für eine robuste Isolierung nicht unbedingt erforderlich ist. Genomweite Analysen haben gezeigt, dass es eine Untergruppe von CBS gibt, die unabhängig von der konvergenten Ausrichtung Chromatinschleifen bilden, wofür ich einen Mechanismus der "Schleifeninterferenz" vorschlage. Weitere Vergleiche ergaben, dass das Niveau der Genexpression von den Abständen zwischen Enhancer und Promoter im linearen Genom abhängen könnte. Durch die Quantifizierung der Isolierung der Grenzen, der Pax3-Fehlexpression und der Schwere der Gliedmaßenfehlbildungen konnte ich schließlich zeigen, dass die TAD-Grenzen die Genexpression und den Phänotyp quantitativ beeinflussen.TAD boundaries are genomic regions with insulator potential located between adjacent chromatin domains, which disruption can cause pathological gene expression. Most TAD boundaries are bound by the CTCF, an architectural protein that forms chromatin loops preferentially between distal pairs of CTCF binding sites (CBSs) with a convergent motif orientation. At TAD boundaries, CBSs are frequently clustered, with motifs displaying a divergent orientation and projecting chromatin loops towards up and downstream regions. How CTCF occupancy modulates insulation at TAD boundaries still remains elusive. Here, I dissected the regulatory logic of CTCF-clustered TAD boundaries by combining genome-wide analysis and in vivo mouse experiments at the Epha4-Pax3 TAD boundary. Analyses of individual deletions revealed a distinct hierarchical contribution of CBS to boundary function. In contrast, combined CBSs deletions revealed a certain degree of functional redundancy and cooperativity between sites. These analyses also demonstrated that the divergent configuration of CBSs, recurrently found at TAD boundaries, is not strictly required for robust insulation. Genome-wide analysis highlighted the existence of a subset of CBSs that establish chromatin loops independently of the convergent orientation bias, for which I propose a mechanism of “loop interference”. This mechanism suggests that CBS forming a robust convergent loop can simultaneously form a non-convergent loop, by stalling Cohesin complexes extruded from both sides. Further comparisons revealed that gene expression levels might depend on enhancer-promoter distances in the linear genome. Finally, by quantifying boundary insulation, Pax3 misexpression and the severity of limb malformation, I demonstrate that TAD boundaries are quantitative modulators of gene expression and phenotypes. Overall, I highlight that TAD boundary composition and strength constitute a fundamental regulatory layer in developmental processes and disease