Genome organization of DNA replication timing and its link to chromatin and transcription

The replication of the genome is a highly organized process. Not every sequence replicates at the same time, instead some genes replicate early, while others replicate later during S phase. The timing of DNA replication is conserved within consecutive cell divisions of a given cell type. The aim of this PhD thesis was a better understanding of the regulation of DNA replication. In particular, I determined the genomic landscape of the timing of DNA replication in the Drosophila genome, and defined the dynamics of replication timing and its connection with chromatin and transcription. Recent genome-wide studies of replication timing and transcription suggested a strong relation between both processes since early replicating genes are more likely to be expressed than genes replicating later during S phase. This correlation is not absolute, therefore raising the question if replication timing is dynamic between different epigenetic states, or if it is static and this correlation is driven mostly by a distinct set of constitutively expressed genes. To create a defined replication timing program, initiation of DNA replication needs to be controlled in space and time. The location and time of firing of the closest origin of replication (ori) defines the replication timing of a certain sequence. However, only few metazoan origins of replication have been identified, and they lack a consensus sequence. Therefore it has been suggested that replication initiation is defined epigenetically. To address this problem I generated datasets for replication timing in two Drosophila cell types representing different developmental states and gender, using high-resolution tiling arrays. This detailed analysis permitted the identification of zones of replication initiation throughout the whole genome. Surprisingly, I could identify a higher number of initiation zones in early and late S phase than in mid S phase. This work also shows that about 20% of the Drosophila genome replicates at different times in the two cell types. These differences in replication timing correlate with differences in gene expression, chromatin modifications and position in the nucleus relative to the nuclear periphery. Interestingly, the dosage compensated male X chromosome replicates predominantly in early S phase. This correlates with chromosome-wide hyperacetylation, often independent of transcription differences. High levels of acetylation on Lysine 16 of Histone H4 were also detected at initiation zones, supporting the model of epigenetically defined replication initiation. In addition, I addressed the potential role of chromatin-bound proteins in modulating replication timing. Using RNA interference, I could show that the absence of Heterochromatin Protein 1 (HP1) has distinct effects on replication timing many of which appear transcription independent. Together, my results reveal organizational principles of DNA replication of the Drosophila genome and indicate that replication timing is dynamic and chromatindependent

RNA sequencing of early round goby embryos reveals that maternal experiences can shape the maternal RNA contribution in a wild vertebrate

It has been proposed that non-genetic inheritance could promote species fitness. Non-genetic inheritance could allow offspring to benefit from the experience of their parents, and could advocate pre-adaptation to prevailing and potentially selective conditions. Indeed, adaptive parental effects have been modeled and observed, but the molecular mechanisms behind them are far from understood. In the present study, we investigated whether maternal RNA can carry information about environmental conditions experienced by the mother in a wild vertebrate. Maternal RNA directs the development of the early embryo in many non-mammalian vertebrates and invertebrates. However, it is not known whether vertebrate maternal RNA integrates information about the parental environment. We sequenced the maternal RNA contribution from a model that we expected to rely on parental effects: the invasive benthic fish species Neogobius melanostomus (Round Goby). We found that maternal RNA expression levels correlated with the water temperature experienced by the mother before oviposition, and identified temperature-responsive gene groups such as core nucleosome components or the microtubule cytoskeleton. Our findings suggest that the maternal RNA contribution may incorporate environmental information. Maternal RNA should therefore be considered a potentially relevant pathway for non-genetic inheritance. Also, the ability of a species to integrate environmental information in the maternal RNA contribution could potentially contribute to species fitness and may also play a role in extraordinary adaptive success stories of invasive species such as the round goby

Evolutionary conservation of the eumetazoan gene regulatory landscape

Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans

Using mass spectrometry imaging to map fluxes quantitatively in the tumor ecosystem

Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, desorption electrospray ionization, and matrix assisted laser desorption ionization reveals alterations in multiple anabolic pathways. De novo fatty acid synthesis flux is increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux is elevated even higher at 8-fold relative to surrounding healthy tissue and highlights the importance of elongase activity in glioma

Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis

Lipid induced stress contributes to metabolic diseases. Here the authors identify small nucleolar RNA 73 (SNORA73) in a screen for genes that protect against lipotoxicity and show that deficiency of SNORA73 reprograms oxidative metabolism and protects against steatohepatitis in mice

Longitudinal metabolomics of human plasma reveals prognostic markers of COVID-19 disease severity

There is an urgent need to identify which COVID-19 patients will develop life-threatening illness so that medical resources can be optimally allocated and rapid treatment can be administered early in the disease course, when clinical management is most effective. To aid in the prognostic classification of disease severity, we perform untargeted metabolomics on plasma from 339 patients, with samples collected at six longitudinal time points. Using the temporal metabolic profiles and machine learning, we build a predictive model of disease severity. We discover that a panel of metabolites measured at the time of study entry successfully determines disease severity. Through analysis of longitudinal samples, we confirm that most of these markers are directly related to disease progression and that their levels return to baseline upon disease recovery. Finally, we validate that these metabolites are also altered in a hamster model of COVID-19

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these “replicon clusters” coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call “replication domains,” separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state

Heterochromatin protein 1 (HP1) modulates replication timing of the Drosophila genome

The replication of a chromosomal region during S phase can be highly dynamic between cell types that differ in transcriptome and epigenome. Early replication timing has been positively correlated with several histone modifications that occur at active genes, while repressive histone modifications mark late replicating regions. This raises the question if chromatin modulates the initiating events of replication. To gain insights into this question, we have studied the function of heterochromatin protein 1 (HP1), which is a reader of repressive methylation at histone H3 lysine 9, in genome-wide organization of replication. Cells with reduced levels of HP1 show an advanced replication timing of centromeric repeats in agreement with the model that repressive chromatin mediates the very late replication of large clusters of constitutive heterochromatin. Surprisingly, however, regions with high levels of interspersed repeats on the chromosomal arms, in particular on chromosome 4 and in pericentromeric regions of chromosome 2, behave differently. Here, loss of HP1 results in delayed replication. The fact that these regions are bound by HP1 suggests a direct effect. Thus while HP1 mediates very late replication of centromeric DNA, it is also required for early replication of euchromatic regions with high levels of repeats. This observation of opposing functions of HP1 suggests a model where HP1-mediated repeat inactivation or replication complex loading on the chromosome arms is required for proper activation of origins of replication that fire early. At the same time, HP1-mediated repression at constitutive heterochromatin is required to ensure replication of centromeric repeats at the end of S phase

Identification of gene regulatory elements in the sea anemone Nematostella vectensis

<p>The genetic complexity, genomic organization, and the level of sequence conservation in Nematostella vectensis, a cnidarian model organism, was found to be more similar to vertebrates than to other bilaterian model organisms. This lead to the assumption that the development of more complex body plans is mainly due to differences in gene regulation, rather than to different gene content between species.</p> <p>Cis-regulatory elements (CREs) (promoters, enhancers or silencers) are essential to regulate gene expression. They have been annotated in the genomes of bilaterian model organisms, but not in a single non-bilaterian metazoan genome.</p> <p>As gene regulatory mechanisms in Nematostella remain unknown, we set out to annotate promoters and enhancers in the Nematostella genome. To this end, we performed chromatin immunoprecipitation (ChIP) of the transcriptional coactivator p300, components of the basal transcription machinery (RNA Pol2) and several histone modifications in different developmental stages followed by Illumina sequencing.</p> <p>Quality of samples was assured and the locations of enrichments are conserved as expected and correlating with gene expression. We employed a supervised learning aproach using ChromHMM for prediction and annotation of putative CREs based on functional chromatin state segments genome-wide. Using this set, we validated some by testing CREs in vivo for their ability to drive expression of a reporter gene (shown in functional disection of Dpp region). The analysis of the different chromatin marks suggests that the genomic location, as well as the function of the different chromatin modifications in regulating gene expression, is conserved between bilaterian model organisms and Nematostella.</p> <p>Our genome-wide map of CREs in a basal metazoan reveals much similarity with bilaterians  and proves usefull in the study of the evolution of gene regulation in animals.</p> <p> </p> <p>Presented at the 2012 EvoNet Symposium in Vienna.</p

Nematostella vectensis transcriptome and gene models v2.0

<p>The gene models, transcripts and other derived data were generated from a comprehensive transcriptome survey of samples taken from five embryonic stages, adult female, and adult male samples. In total 1.6 billion 76-100 bp pair-end reads were obtained. The reads were mapped onto the N. vectensis genome v1.0, and new gene models were constructed using the RNA-seq evidence with the Augustus gene caller. These RNA-seq evidence based gene models are identified by a NVE prefix.</p