Epigenetic regulation of transcription and genome evolution in Verticillium dahliae

Through the association of protein complexes to DNA, the nuclear genome is broadly organized into accessible euchromatin and condensed heterochromatin. Chemical and physical alterations to these types of chromatin may impact their organization and functionality, and are therefore important regulators of nuclear processes in eukaryotes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced effector genes that are important for pathogenicity. Chapter 1 of this thesis introduces interactions between plants and microbial pathogens, with a particular focus on the plant pathogenic Ascomycete fungus Verticillium dahliae; the subject of study of this thesis research. V. dahliae is a soil-borne filamentous fungus that can infect hundreds of host plants and colonizes their xylem vessels, leading to wilt diseases that can devastate crop yields. Chapter 1 outlines a prevalent hypothesis on epigenetic regulation of effector gene expression, stating that chromatin at effector gene-containing genomic regions is condensed when the fungus does not grow inside its plant hosts. Consequently, in order to express effectors in planta, the pathogen requires to chemically alter its chromatin, leading to chromatin de-condensation.In a similar fashion as has been reported for the genomes of various other fungal species, the genome of V. dahliae can be characterized as a so-called two-speed genome, in which particular regions are more plastic, and evolve more rapidly, than the evolutionary more stable core genome. In Chapter 2, we explore epigenome features, including DNA methylation, chromatin accessibility and histone methylation, and show that the plastic genome of V. dahliae is associated with tri-methylation of lysine 27 on histone 3 (H3K27me3) and with accessible chromatin. Using a machine learning approach trained on chromatin, and validated through orthogonal analyses, we now identified approximately twice as much DNA in plastic regions than previously recognized. The collective plastic regions are now referred to as adaptive genomic regions (AGRs). Our results show that a specific chromatin profile defines the plastic genome, and highlight how different epigenetic factors contribute to the organization of AGRs.H3K27me3 is generally associated with facultative heterochromatin, which represents a closed conformation of the DNA and corresponding inaccessibility to the transcriptional machinery, yet can de-condense upon recognition of external cues. In Chapter 3, we investigated the involvement of H3K27me3 in transcriptional regulation by comparing H3K27me3 coverage and transcription for V. dahliae cultivated in three in vitro cultivation media. We show that although various genes in AGRs are differentially expressed between the cultivation media, H3K27me3 domains globally display stable profiles. However, we do observe local quantitative differences in H3K27me3 coverage that associate with differentially expressed genes, although this is not a ubiquitous pattern. Overall, our results demonstrate that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such dynamics. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it is not a conditionally responsive global regulator of differential transcription. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms, or may serve additional genomic regulatory functions.Methylation of cytosine nucleobases (5-methylcytosine, 5mC) is an important epigenetic control mechanism that is restricted to genomic regions containing transposable elements (TEs) in many organisms, including fungi. Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species that contain both genes. In Chapter 4, we use functional analyses to show that Dim2, but not Dnmt5 or the putative sexual cycle-related DNA methyltransferase Rid, is responsible for the majority of DNA methylation in V. dahliae. Single and double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or TEs. In contrast, Hp1 and Dim5 mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display transcriptional reprogramming in AGRs. As these AGRs are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. Overall, our results suggest that Dim2 is the main DNA methyltransferase in V. dahliae and, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective of Dnmt5 presence. We speculate that Dnmt5 and Rid act under specific, presently enigmatic, conditions or, alternatively, act in DNA-associated processes other than DNA methylation.Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between, and even within, species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In Chapter 5, we identified the centromeres in each of the ten species of the Verticillium genus and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (~150 kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in V. dahliae. Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated LTR retrotransposon is strongly associated with centromeric regions in some Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, this chapter highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the Verticillium genus.The three dimensional (3D) folding of DNA in the nucleus organizes chromosomes into so-called topologically associating domains (TADs). These TADs are self-interacting genomic regions that display less interaction with adjacent regions. Functionally, TADs have been implicated in transcriptional regulation as well as in genome evolution in numerous organisms, yet in fungi the functional implication of these regions remains less clear. In Chapter 6, we utilize Hi-C data generated for V. dahliae to investigate TAD organization and its influence on transcription. Additionally, we compare the TAD organization between two V. dahliae strains as well as with other Verticillium species to study the conservation of TADs throughout the genus. Remarkably, we find that TADs in the AGRs of V. dahliae are less well insulated than TADs in the core genome, indicating that TADs in AGRs are not as well established as those in the core genome. Moreover, TADs in AGRs display significantly more co-regulation of gene expression than TADs in the core genome. Furthermore, genes located in TAD boundaries, i.e. regions that delineate adjacent TADs, in AGRs are generally lower expressed in vitro, while stronger differentially expressed between in vitro conditions, than genes located in TADs in AGRs. We find that TAD boundaries are depleted for structural variation between Verticillium species, and that TADs are generally conserved in the Verticillium genus. Overall, our study points towards an association between TAD organization and transcriptional regulation as well as genome evolution in Verticillium.Finally, Chapter 7 revisits the prevalent hypothesis on epigenetic regulation of effector gene expression through extensive chromatin dynamics, as presented in Chapter 1. I conclude that this hypothesis is likely too simple, and therefore I bring forward alternative hypotheses to explain the potential role of H3K27me3 in transcriptional regulation of in planta and in vitro differentially expressed genes. Furthermore, the implications of the findings presented in this thesis, regarding epigenetic mechanisms and spatial genome organization, are discussed in the broader context of nuclear processes in eukaryotic organisms