Stochastic modeling of DNA demethylation dynamics in ESCs

DNA methylation and demethylation are opposing processes that when in balance create stable patterns of epigenetic memory. The control of DNA methylation pattern formation in replication dependent and independent demethylation processes has been suggested to be influenced by Tet mediated oxidation of a methylated cytosine, 5mC, to a hydroxylated cytosine, 5hmC. Based only on in vitro experiments, several alternative mechanisms have been proposed on how 5hmC influences replication dependent maintenance of DNA methylation and replication independent processes of active demethylation. In this thesis we design an extended and easily generalizable hidden Markov model that uses as input hairpin (oxidative-)bisulfite sequencing data to precisely determine the over time dynamics of 5mC and 5hmC, as well as to infer the activities of the involved enzymes at a single CpG resolution. Developing the appropriate statistical and computational tools, we apply the model to discrete high-depth sequenced genomic loci, and on a whole genome scale with a much smaller sequencing depth. Performing the analysis of the model’s output on mESCs data, we show that the presence of Tet enzymes and 5hmC has a very strong impact on replication dependent demethylation by establishing a passive demethylation mechanism, implicitly impairing methylation maintenance, but also down-regulating the de novo methylation activity.DNA-Methylierung und Demethylierung sind gegenläufige Prozesse, die im Gleichgewicht stabile Muster des epigenetischen Gedächtnisses erzeugen. Es wird angenommen, dass die Kontrolle der DNA-Methylierungsmusterbildung in replikationsabhängige und unabhängige Demethylierungsprozesse durch Tet-regulierte Oxidation eines methylierten Zytosins (5mC) zu einem hydroxylierten Zytosin (5hmC) beeinflusst wird. Aufgrund von In-Vitro-Experimenten, wurden verschiedene Mechanismen vorgeschlagen wie 5hmC die replikationsabhängige Aufrechterhaltung der DNA-Methylierung und die replikationsunabhängigen Prozesse der aktiven Demethylierung beeinflusst. In dieser Arbeit entwerfen wir ein erweitertes und leicht verallgemeinertes Hidden Markov Modell, das mit Hilfe von Hairpin (oxidative-)Bisulfit Sequenzierung gewonnener Daten die Zeitdynamik von 5mC und 5hmC genau bestimmt und die Aktivitäten der beteiligten Enzyme auf der Ebene einzelner CpGs scha ̈tzt. Wir entwickeln geeignete statistische Methoden, um das Modell sowohl auf der Ebene der sequenzspezifischen Tiefensequenzierung einzelner Loci, als auch auf genomweiter Ebene mit stark verringerter Sequenzierungstiefe anzuwenden. Wir zeigen, dass die Anwesenheit von Tet-Enzymen und 5hmC einen sehr starken Einfluss auf die replikationsabhängige Demethylierung hat, indem sie einen passiven Demethylierungsmechanismus etabliert, der die Methylierungserhaltung implizit beeinträchtigt, aber auch die de novo-Methylierung herunterreguliert

Targeting and dynamics of gene repression during stem cell differentiation

The identity and function of different cellular subtypes critically depend on their unique set of expressed genes. Gene expression programs and their changes during development are mainly controlled by sequence-specific DNA binding factors. It has recently become clear that chromatin modifications are important regulators of these processes. While there are several chromatin-based pathways that correlate with gene repression, their exact role in silencing remains elusive. Moreover, for many repressive chromatin modifications a complete picture of the genomic distribution and its dynamics during development is lacking. Finally, it is still unclear how these genomic patterns of repressive chromatin marks are established. For my PhD work, I set out to address these questions by studying the targeting of H3K9me2 and DNA methylation during cellular differentiation. Our analysis revealed that H3K9me2 is highly abundant in embryonic stem cells and occurs in large domains that occupy more than half of the genome. H3K9me2 marks chromatin outside of transcribed, active or polycomb regulated sites, possibly keeping it in a repressed state. Importantly, abundance of H3K9me2 increases only slightly during neuronal differentiation, with a localized gain occurring at gene bodies of transcribed genes. By gene expression profiling we further show that the transcriptome complexity is very similar in stem cells and derived post-mitotic neurons. These data are in contrast to a previously suggested model which states that the pluripotent state of stem cells is accompanied by a global reduction in heterochromatin and a concomitant higher proportion of transcription. Together with results from other groups our data rather indicate that repressive chromatin is abundant in stem cells and upon differentiation gets redistributed only locally and not globally. It has been suggested that such a localized increase of repression at gene regulatory regions helps stabilizing lineage choices and differentiation processes. In order to investigate how chromatin-based repression pathways are targeted to gene regulatory sites, we focused on DNA methylation, a modification whose catalysis and epigenetic propagation are well understood. By site-specific sequence integration experiments we show that 1 kb promoter elements are sufficient to recapitulate endogenous DNA methylation patterns in stem cells and their dynamic changes upon differentiation, in a process that is independent of transcription. In stem cells, promoters are protected from DNA methylation by small sequence elements that we termed methylation determining regions (MDRs). Protection from DNA methylation by MDRs depends on a combination of DNA binding motifs, which get recognized by transcription factors such as RFX2. It has been speculated before that establishment of an unmethylated promoter state is facilitated by proteins that recognize unmethylated CpGs. While not excluding a role in maintenance, our data suggest that CpG-richness alone is not sufficient for initiation of this chromatin state. Remarkably, no additional sequence besides an MDR is needed to recapitulate differentiation-induced de novo methylation. Moreover, MDRs are able to protect neighboring sequences from DNA methylation in stem cells and from de novo methylation during differentiation. These results imply that one possible way of differentiation-induced de novo methylation could involve reduced binding of factors that protect from DNA methylation. In summary, H3K9me2 and DNA methylation occupy per default most the genome, even in cells with a high developmental potential. Accordingly, cellular differentiation is accompanied by focal, rather than global changes in repressive chromatin modifications. In the case of DNA methylation, such local changes at gene regulatory sites are determined by the underlying sequence and likely involve binding of transcription factors that protect from DNA methylation

Molecular mapping of nuclear organization in the mouse preimplantation embryo

Upon fertilization the two parental genomes are extensively reprogrammed to give rise to a totipotent state. In the mammalian embryo, this epigenetic reprogramming involves an extensive three dimensional (3D) rearrangement of nuclear organization which only recently have been started to be investigated on a genome-wide scale. The positioning of loci relative to the nuclear periphery has been shown to change during differentiation, potentially regulating gene expression and chromatin. Therefore, it is of question whether the nuclear reorganization in early embryonic cells correlates with or even regulates genome function and embryo development. During my PhD work, we have created maps of lamina associated domains (LADs) from mouse preimplantation embryos and oocytes at the single cell level. LADs are genomic regions that reside at the nuclear periphery and represent a lowly transcribed, gene-poor fraction of the genome originally identified in somatic cells. We have found that LADs are absent in oocytes but become established already in zygotes and are dynamically rearranged during the 2- and 8-cell stages with little heterogeneity between individual cells. We obtained LAD data from hybrid embryos to distinguish the parental genomes by single nucleotide polymorphisms (SNPs) in sequencing. Our analysis unravelled differences in genome organization between the two parental alleles that likely reflect their different germline history. Moreover, we find that LAD formation precedes the maturation of topologically associated domains (TADs) in a DNA replication independent manner. Additionally, we observed that only the X chromosome contacts the lamina in oocytes, potentially through an interaction with the Lamin B Receptor (LBR) protein. Eventually, we identified an epigenetic asymmetry of H3K4 methylation on LADs between the paternal and maternal genomes in zygotes. We found that the experimental reduction of the H3K4me3 histone mark by the overexpression of the lysine demethylase Kdm5b results in a loss of LAD structure, specifically in the paternal zygotic genome. In conclusion, we have uncovered a novel mechanism of allele specific LAD formation through histone methylation. Additionally, this work provides genome wide information on mouse preimplantation nuclear organization contributing a resource for further epigenetic studies of early embryos

Hybrid Decay: A Transgenerational Epigenetic Decline in Vigor and Viability Triggered in Backcross Populations of Teosinte with Maize.

In the course of generating populations of maize with teosinte chromosomal introgressions, an unusual sickly plant phenotype was noted in individuals from crosses with two teosinte accessions collected near Valle de Bravo, Mexico. The plants of these Bravo teosinte accessions appear phenotypically normal themselves and the F1 plants appear similar to typical maize × teosinte F1s. However, upon backcrossing to maize, the BC1 and subsequent generations display a number of detrimental characteristics including shorter stature, reduced seed set, and abnormal floral structures. This phenomenon is observed in all BC individuals and there is no chromosomal segment linked to the sickly plant phenotype in advanced backcross generations. Once the sickly phenotype appears in a lineage, normal plants are never again recovered by continued backcrossing to the normal maize parent. Whole-genome shotgun sequencing reveals a small number of genomic sequences, some with homology to transposable elements, that have increased in copy number in the backcross populations. Transcriptome analysis of seedlings, which do not have striking phenotypic abnormalities, identified segments of 18 maize genes that exhibit increased expression in sickly plants. A de novo assembly of transcripts present in plants exhibiting the sickly phenotype identified a set of 59 upregulated novel transcripts. These transcripts include some examples with sequence similarity to transposable elements and other sequences present in the recurrent maize parent (W22) genome as well as novel sequences not present in the W22 genome. Genome-wide profiles of gene expression, DNA methylation, and small RNAs are similar between sickly plants and normal controls, although a few upregulated transcripts and transposable elements are associated with altered small RNA or methylation profiles. This study documents hybrid incompatibility and genome instability triggered by the backcrossing of Bravo teosinte with maize. We name this phenomenon "hybrid decay" and present ideas on the mechanism that may underlie it

Strategies for analyzing bisulfite sequencing data

DNA methylation is one of the main epigenetic modifications in the eukaryotic genome and has been shown to play a role in cell-type specific regulation of gene expression, and therefore cell-type identity. Bisulfite sequencing is the gold-standard for measuring methylation over the genomes of interest. Here, we review several techniques used for the analysis of high-throughput bisulfite sequencing. We introduce specialized short-read alignment techniques as well as pre/post-alignment quality check methods to ensure data quality. Furthermore, we discuss subsequent analysis steps after alignment. We introduce various differential methylation methods and compare their performance using simulated and real bisulfite-sequencing datasets. We also discuss the methods used to segment methylomes in order to pinpoint regulatory regions. We introduce annotation methods that can be used further classification of regions returned by segmentation or differential methylation methods. Lastly, we review software packages that implement strategies to efficiently deal with large bisulfite sequencing datasets locally and also discuss online analysis workflows that do not require any prior programming skills. The analysis strategies described in this review will guide researchers at any level to the best practices of bisulfite sequencing analysis

HOT or not: Examining the basis of high-occupancy target regions

High-occupancy target (HOT) regions are the segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and the associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solemnly defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed “hyper-ChIPable” regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpG-rich k-mers and enrichment of RNA-DNA hybrids (R-loops) and DNA tertiary structures (G-quadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies