Three-dimensional Folding of Eukaryotic Genomes

Chromatin packages eukaryotic genomes via a hierarchical series of folding steps, encrypting multiple layers of epigenetic information, which are capable of regulating nuclear transactions in response to complex signals in environment. Besides the 1-dimensinal chromatin landscape such as nucleosome positioning and histone modifications, little is known about the secondary chromatin structures and their functional consequences related to transcriptional regulation and DNA replication. The family of chromosomal conformation capture (3C) assays has revolutionized our understanding of large-scale chromosome folding with the ability to measure relative interaction probability between genomic loci in vivo. However, the suboptimal resolution of the typical 3C techniques leaves the levels of nucleosome interactions or 30 nm structures inaccessible, and also restricts their applicability to study gene level of chromatin folding in small genome organisms such as yeasts, worm, and plants. To uncover the “blind spot” of chromatin organization, I developed an innovative method called Micro-C and an improved protocol, Micro-C XL, which enable to map chromatin structures at all range of scale from single nucleosome to the entire genome. Several fine-scale aspects of chromatin folding in budding and fission yeasts have been identified by Micro-C, including histone tail-mediated tri-/tetra-nucleosome stackings, gene crumples/globules, and chromosomally-interacting domains (CIDs). CIDs are spatially demarcated by the boundaries, which are colocalized with the promoters of actively transcribed genes and histone marks for active transcription or turnover. The levels of chromatin compaction are regulated via transcription-dependent or transcription-independent manner – either the perturbations of transcription or the mutations of chromatin regulators strongly affect the global chromatin folding. Taken together, Micro-C further reveals chromatin folding behaviors below the sub-kilobase scale and opens an avenue to study chromatin organization in many biological systems

The genetic architecture of DNA replication timing in human pluripotent stem cells.

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation

Bioinformatic analyses of the structural and functional complexity in chromosomal interactomes

Evolution requires information storage systems with different demands with respect to persistence. While the genome provides a mechanism for long term, static and accurate information storage, it is incapable of mediating adaptation to short term changes in the environment. Chromatin, however, constitutes a dynamic, reprogrammable memory with different levels of persistence. Moreover, chromatin states carry information not only in 2D, i.e. in the structure of the primary chromatin fibre, but also in the 3D organization of the genome in the nuclear space. The following thesis delves into the new bioinformatic and wet lab protocols developed to map, quantitative and functionally analyze the 3D architecture of chromatin. The chromatin insulator protein CTCF is a major factor underlying the 3D organization of the epigenome. We have uncovered, however, that CTCF binding sites within a regulatory region have multiple functions that are influenced by the chromatin environment and possibly the combinatorial usage of the 11 Zn-fingers of CTCF (Paper I). This observation exemplifies that understanding the function of dynamic and transient chromatin fibre interactions requires novel technology that enables the detection of 3D chromatin folding with high resolution in single cells and in small cell populations. We therefore set out to devise a novel method for the visualization of higher order chromatin structures by combining the strengths of both DNA Fluorescent In Situ Hybridization (FISH) and In Situ Proximity Ligation Assay (ISPLA) technologies (Paper II). The resulting Chromatin in Situ Proximity (ChrISP) assay thus takes advantage of the direct contact detection of ISPLA and the locus-specific nature of FISH and uncovered the existence of compact chromatin structures at the nuclear envelope with unprecedented resolution. To complement ChrISP with a high throughput method capable of quantitatively recovering chromatin fibre contacts in small cell populations, we furthermore innovated the Nodewalk assay (Paper III). The protocol builds on existing ligation based chromosome conformation capture methods, but features significant reduction in the random ligation event frequency, inclusion of negative and positive ligation controls, iterative template resampling, increased signal to noise ratio and improved sensitivity. Using this technique, we have uncovered a cancer cell-specific, productive chromatin fibre interactome connecting the promoter and enhancer of c-MYC to a network of enhancers and super-enhancers. Underpinning this new protocol, I have developed the Nodewalk Analysis Pipeline (NAP) (Paper IV). This suite of tools consists of preprocessing, analysis and post-processing modules designed specifically for the rapid and efficient analysis of Nodewalk datasets through an interactive and user-friendly web based interface. Overall the work described in this thesis advances our understanding of the role of CTCF in nuclear organization and provides innovative wet lab techniques along with specialized software tools. Moreover, this work is an example of an emerging trend where the challenge of understanding chromatin dynamics within the 3D nuclear architecture demands a close synergistic collaboration between the fields of biology, biotechnology and bioinformatics

Epigenetic regulation of DNA replication in Drosophila melanogaster

How origins of DNA replication are specified and activated in the context of an intact metazoan genome remains poorly understood. In contrast to Saccharomyces cerevisiae, replication initiation in metazoan genomes is not directed by well-defined sequence motifs. Rather, local chromatin environments have emerged as potential regulators of replication, yielding early and late replicating regions of the genome. Transcriptionally active, accessible euchromatin typically replicates early during S phase, whereas transcriptionally repressive, inaccessible heterochromatin typically replicates late. Current models of replication posit a stochastic process in which a higher density of specified origins in euchromatin compared to heterochromatin increases the probability of replication initiation, resulting in the earlier replication of euchromatin relative to heterochromatin. Despite strong genome-wide correlations between replication and chromatin, a true causal relationship between the two has yet to be determined. We investigated how chromatin organization impacts replication in Drosophila using our genetic platform in which endogenous histone genes are replaced with transgenic histone genes encoding mutations that prevent modification of specific histone residues. To explore the relationship between euchromatin and replication, we implemented a whole-genome sequencing method to produce genome-wide replication timing profiles. We analyzed the X Chromosome, which in Drosophila is 2-fold more transcriptionally active, replicates earlier, and is hyper-acetylated at H4K16 in XY males relative to XX females. H4K16R mutation prevents transcriptional hyper-activation and earlier replication of the male X chromosome, consistent with the notion that transcription promotes early replication. To determine whether perturbation of heterochromatin affects late replication, we generated replication profiles from H3K9R mutant tissue. Despite well-known correlations between late replication and heterochromatin, perturbation of heterochromatin structure through H3K9R mutation does not result in large-scale changes in replication timing suggesting critical regulation beyond chromatin structure. To identify other contributors to replication timing control, we explored the relative contributions of cell lineage, cell cycle, and the trans-acting factor Rif1. We identified that cell lineage, rather than changes in cell cycle status, drive replication timing programs. Furthermore, Rif1 regulates replication timing in a tissue-specific manner supporting the notion that additional mechanisms beyond chromatin structure are key regulators of replication of metazoan genomes.Doctor of Philosoph

Investigating the 3D chromatin architecture with fluorescence microscopy

Chromatin is an assembly of DNA and nuclear proteins, which on the one hand has the function to properly store the 2 meters of DNA of a diploid human nucleus in a small volume and on the other hand regulates the accessibility of specific DNA segments for proteins. Many cellular processes like gene expression and DNA repair are affected by the three-dimensional architecture of chromatin. Cohesin is an important and well-studied protein that affects three-dimensional chromatin organization. One of the functions of this motor protein is the active generation of specific domain structures (topologically associating domains (TADs)) by the process of loop extrusion. Studies of cohesin depleted cells showed that TAD structures were lost on a population average. Due to this finding, the question arose, to what extent the functional nuclear architecture, that can be detected by confocal and structured illumination microscopy, is impaired when cells were cohesin depleted. The work presented in this thesis could show that the structuring of the nucleus in areas with different chromatin densities including the localization of important nuclear proteins as well as replication patterns was retained. Interestingly, cohesin depleted cells proceeded through an endomitosis leading to the formation of multilobulated nuclei. Obviously, important structural features of chromatin can form even in the absence of cohesin. In the here presented work, fluorescence microscopic methods were used throughout, and an innovative technique was developed, that allows flexible labeling of proteins with different fluorophores in fixed cells. With this technique DNA as well as peptide nucleic acid (PNA) oligonucleotides can be site-specifically coupled to antibodies via the Tub-tag technology and visualized by complementary fluorescently labeled oligonucleotides. The advantages and disadvantages of PNAs as docking strands are discussed in this thesis as well as the use of PNAs in fluorescence in situ hybridization (FISH). In the next study, which is part of this work, a combination of FISH and super-resolution microscopy was used. There it could be shown that DNA segments of 5 kb can form both compact and elongated configurations in regulatory active as well as inactive chromatin. Coarse-grained modeling of these microscopic data, in agreement with published data from other groups, has suggested that elongated configurations occur more frequently in DNA segments in which the occupancy of nucleosomes is reduced. The microscopically measured distance distributions could only be simulated with models that assume different densities of nucleosomes in the population. Another result of this study was that inactive chromatin - as expected - shows a high level of compaction, which can hardly be explained with common coarse-grained models. It is possible that environmental effects that are difficult to simulate play a role here. Chromatin is a highly dynamic structure, and its architecture is constantly changing, be it through active processes such as the effect of cohesin investigated here or through thermodynamic interactions of nucleosomes as they are simulated in coarse-grained models. It will take a long time until we adequately understand these dynamic processes and their interplay

CaTCHing the functional and structural properties of chromosome folding

Proper development requires that genes are expressed at the right time, in the right tissue, and at the right transcriptional level. In metazoans, this involves long-range cis-regulatory elements such as enhancers, which can be located up to hundreds of kilobases away from their target promoters. How enhancers find their target genes and avoid aberrant interactions with non-target genes is currently under intense investigations. The predominant model for enhancer function involves its direct physical looping between the enhancer and target promoter. The three-dimensional organization of chromatin, which accommodates promoter- enhancer interactions, therefore might play an important role in the specificity of these interactions. In the last decade, the development of a class of techniques called chromosome conformation capture (3C) and its derivatives have revolutionized the field of chromatin folding. In particular, the genome-wide version of 3C, Hi-C, revealed that mammalian chromosomes possess a rich hierarchy of folding layers, from multi-megabase compartments corresponding to mutually exclusive associations of active and inactive chromatin to topologically associating domains (TADs), which reflect regions with preferential internal interactions. Although the mechanisms that give rise to this hierarchy are still poorly understood, there is increasing evidence to suggest that TADs represent fundamental functional units for establishing the correct pattern of enhancer-promoter interactions. This is thought to occur through two complementary mechanisms: on the one hand, TADs are thought to increase the chances that regulatory elements meet each other by confining them within the same domain; on the other hand, by segregation of physical interactions across the boundary to avoid unwanted events to occur frequently. It is however unclear whether the properties that have been attributed to TADs are specific to TADs, or rather common features among the whole hierarchy. To address this question, I have implemented an algorithm named Caller of Topological Chromosomal Hierarchies (CaTCH). CaTCH is able to detect nested hierarchies of domains, allowing a comprehensive analysis of structural and functional properties across the folding hierarchy. By applying CaTCH to published Hi-C data in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs), I showed that TADs emerge as a functionally privileged scale. In particular, TADs appear to be the scale where accumulation of CTCF at domain boundaries and transcriptional co-regulation during differentiation is maximal. Moreover, TADs appear to be the folding scale where the partitioning of interactions within transcriptionally active domains (and notably between active enhancers and promoters) is optimized. 3C-based methods have enabled fundamental discoveries such as the existence of TADs and CTCF-mediated chromatin loops. 3C methods detect chromatin interactions as ligation products after crosslinking the DNA. Crosslinking and ligation have been often criticized as potential sources of experimental biases, raising the question of whether TADs and CTCF- mediated chromatin loops actually exist in living cells. To address this, in collaboration with Josef Redolfi, we developed a new method termed ‘DamC’ which combines DNA methylation with physical modeling to detect chromosomal interactions in living cells, at the molecular scale, without relying on crosslinking and ligation. By applying DamC to mouse ESCs, we provide the first in vivo and crosslinking- and ligation-free validation of chromosomal structures detected by 3C-methods, namely TADs and CTCF-mediated chromatin loops. DamC, together with 3C-based methods, thus have shown that mammalian chromosomes possess a rich hierarchy of folding layers. An important challenge in the field is to understand the mechanisms that drive the establishment these folding layers. In this sense, polymer physics represent a powerful tool to gain mechanistic insights into the hierarchical folding of mammalian chromosomes. In polymer models, the scaling of contact probability, i.e. the contact probability as a function of genomic distance, has been often used to benchmark polymer simulations and test alternative models. However, the scaling of contact probability is only one of the many properties that characterize polymer models raising the question of whether it would be enough to discriminate alternative polymer models. To address this, I have built finite-size heteropolymer models characterized by random interactions. I showed that finite-size effects, together with the heterogeneity of the interactions, are sufficient to reproduce the observed range of scaling of contact probability. This suggests that one should be careful in discriminating polymer models of chromatin folding based solely on the scaling. In conclusion, my findings have contributed to achieve a better understanding of chromatin folding, which is essential to really understand how enhancers act on promoters. The comprehensive analyses using CaTCH have provided conceptually new insights into how the architectural functionality of TADs may be established. My work on heteropolymer models has highlighted the fact that one should be careful in using solely scaling to discriminate physical models for chromatin folding. Finally, the ability to detect TADs and chromatin loops using DamC represents a fundamental result since it provides the first orthogonal in vivo validation of chromosomal structures that had essentially relied on a single technology

Investigating 3D genome organisation in Caenorhabditis elegans with Accessible Region Conformation Capture (ARC-C)

3C and its derivatives have been applied in various organisms to study chromatin architecture. However, these methods have limitations: most of them are limited to restriction-fragment resolution and all of them, with the exception of Hi-C, only survey a pre-defined subset of the genome. I developed a variant of Hi-C, named Accessible Region Conformation Capture (ARC-C), in C. elegans, which interrogates genome organisation at multiple scales genome-wide - from domains and compartments to high resolution interactions between regulatory elements. I applied ARC-C in wild-type Bristol N2, met-2 set-25 mutants that have no H3K9 methylation to study the effects on domain and compartment formation. In these mutants, compartmentalisation (i.e. inter-domain interactions) between H3K27me3-enriched regulated domains is reduced. I also used ARC-C in blmp-1 mutants to understand the role of BLMP-1 in chromatin looping. In blmp-1 mutants, interactions between putative BLMP-1 mediated loops for downregulated genes are significantly reduced. In wild-type worms, when surveying significant interactions at 500 bp resolution, I observe the presence of dense clusters of significant interactions anchored at high occupancy target (HOT) regions that I call “hubs.” Interestingly, the deletion of these hubs does not affect the transcription of linked or local genes. However, local interactions are altered and some extent of redundancy is observed. To improve our ARC-C protocol, I tested several variations with different enzymes and biotin-mediated streptavidin beads pulldown. In all, ARC-C revealed insights into genome organisation in C. elegans and I have made progress toward a next-generation version of the method.A*STAR National Science Scholarship (PhD