Histone lysine methylation in the context of nuclear architecture

The impact of histone lysine methylation as an essential epigenetic mechanismn for gene regulation has been demonstrated by numerous studies where it was functionally and structurally linked to euchromatin and heterochromatin.In this work the 3D architecture and spatial interrealtionships of different histone lysine methylation sites was investigated in various human cell types

Study of the role of plant nuclear envelope and lamina-like components in nuclear and chromatin organisation using 3D imaging

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The fi aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphol- ogy and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with diff t nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromo- centres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC com- plex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes ad- vantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional NE. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes

Transcriptional and post-transcriptional regulation of leaf development in Arabidopsis thaliana

Plant growth follows a strict developmental program but needs to incorporate also environmental cues to adapt to the encountered conditions. This requires a complex regulatory network to ensure an appropriate response to changing conditions. We used the first leaf pair of Arabidopsis thaliana as a model system to study the regulation of organ development. Leaf growth can be divided in subsequent phases according to the major process driving it. In a young leaf primordium cells divide continuously and cell size homeostasis is ensured by matching rates of cell expansion. Next, cell division ceases and cell expansion becomes the driving force for growth. When the leaf has attained its final size, maturity is reached. In this thesis, I studied the regulation of leaf development at two regulatory levels. At the gene level, we analyzed the function of the CYCA2 core cell cycle regulatory gene family. We also studied the function of two new proliferation specific gene families putatively involved in cell cycle regulation. On the other hand, we profiled small RNA sequences during development and linked this with the occurrence of DNA methylation. The core machinery of the cell cycle in plants has been thoroughly studied, but our knowledge on how developmental and environmental signals impinge on cell division is still limited. CYCA2s are known core cell cycle regulators, involved in G2-to-M transition. Here, we studied the functional requirement of this gene family and showed that transcriptional repression is required for specific differentiation processes. Members of the CYCA2 protein family function in vascular development and differentiation of guard cells. For the latter process, we demonstrated that FOUR LIPS and MYB88, two transcription factors involved in stomatal development, directly repress CYCA2;3 expression, thus ensuring correct guard cell differentiation. Next to known ‘core’ cell cycle regulating genes, we also selected proliferation specific genes with unknown function, assuming them to be involved in the cell division process. We focused on two small gene families: three genes with four transmembrane domains (4TMs) and two genes containing three High Mobility Group (HMG) domains (3xHMG-box). Expression analysis and localization of transcriptional fusions with a fluorescent marker confirmed for both gene families the highly proliferation-specific expression pattern. Moreover, both families are highly induced in the M-phase of the cell cycle in synchronized cell cultures. The 4TMs localize to the cell plate during mitosis and we observed defects in cell plate formax tion upon overexpression and depletion of these genes. Therefore, we hypothesize that the 4TM genes are involved in formation of the cell plate. Profiling of small RNAs (sRNAs) in plants has thusfar mainly been focused on inflorescence tissue or whole seedlings. Here, we studied sRNAs during the different phases of development. Early in development, microRNAs implicated in nutrient stress response are upregulated, suggesting that at this phase nutrient availability is limiting for growth. We showed that specifically 24-nt sRNAs increase in expression during development. This class of sRNAs is known to be involved in RNA-dependent DNA methylation (RdDM) and can thus silence both transposons and genes. In general, the expression of sRNAs matching the coding sequences of protein-coding genes is positively correlated to the mRNA expression of this gene. We specifically selected genes that do not show this correlation, which were highly enriched in two categories: targets of microRNAs and trans-acting siRNAs, which generate phased sRNAs upon cleavage, and genes for which the sRNA profile is enriched for 24-nt sRNAs. This latter category is likely regulated through RdDM as this subset of genes shows increased DNA methylation in the gene body. This suggests that sRNA regulation could play an important role in regulating the leaf developmental process not only by preserving genome integrity by repressing transposon activity but also through silencing of protein-coding genes

Washington University Senior Undergraduate Research Digest (WUURD), Spring 2018

From the Washington University Office of Undergraduate Research Digest (WUURD), Vol. 13, 05-01-2018. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Scienc

3D Organization of Eukaryotic and Prokaryotic Genomes

There is a complex mutual interplay between three-dimensional (3D) genome organization and cellular activities in bacteria and eukaryotes. The aim of this thesis is to investigate such structure-function relationships. A main part of this thesis deals with the study of the three-dimensional genome organization using novel techniques for detecting genome-wide contacts using next-generation sequencing. These so called chromatin conformation capture-based methods, such as 5C and Hi-C, give deep insights into the architecture of the genome inside the nucleus, even on a small scale. We shed light on the question how the vastly increasing Hi-C data can generate new insights about the way the genome is organized in 3D. To this end, we first present the typical Hi-C data processing workflow to obtain Hi-C contact maps and show potential pitfalls in the interpretation of such contact maps using our own data pipeline and publicly available Hi-C data sets. Subsequently, we focus on approaches to modeling 3D genome organization based on contact maps. In this context, a computational tool was developed which interactively visualizes contact maps alongside complementary genomic data tracks. Inspired by machine learning with the help of probabilistic graphical models, we developed a tool that detects the compartmentalization structure within contact maps on multiple scales. In a further project, we propose and test one possible mechanism for the observed compartmentalization within contact maps of genomes across multiple species: Dynamic formation of loops within domains. In the context of 3D organization of bacterial chromosomes, we present the first direct evidence for global restructuring by long-range interactions of a DNA binding protein. Using Hi-C and live cell imaging of DNA loci, we show that the DNA binding protein Rok forms insulator-like complexes looping the B. subtilis genome over large distances. This biological mechanism agrees with our model based on dynamic formation of loops affecting domain formation in eukaryotic genomes. We further investigate the spatial segregation of the E. coli chromosome during cell division. In particular, we are interested in the positioning of the chromosomal replication origin region based on its interaction with the protein complex MukBEF. We tackle the problem using a combined approach of stochastic and polymer simulations. Last but not least, we develop a completely new methodology to analyze single molecule localization microscopy images based on topological data analysis. By using this new approach in the analysis of irradiated cells, we are able to show that the topology of repair foci can be categorized depending the distance to heterochromatin

Interplay of genetic, epigenetic and transcription factors in the regulation of transcriptional variation in Plasmodium falciparum

[eng] The most severe form of malaria, caused by Plasmodium falciparum parasites, still kills over half a million people every year, most of them children under the age of five. Despite huge research efforts, reduction in the global burden of disease has stalled in recent years. P. falciparum has a very complex life cycle including, among other steps, sexual reproduction in female Anopheles mosquitos and an asexual intra-erythoricitic development cycle (IDC) inside the human host, which causes the disease. During the IDC, the parasite needs to continuously adapt to changes in its environment including fluctuations in blood temperature, concentration of nutrients and other metabolites, presence of drugs, and a constant fight against the host’s immune system. In this thesis, we have studied the adaptation mechanisms of P. falciparum to this plethora of challenges, with a special focus on clonally variant genes (CVGs). In P. falciparum, CVGs are a set of genes, participating in host-parasite interactions, which can be found both in a transcriptionally active state, characterized by euchromatin, or a transcriptionally silenced state, characterized by heterochromatin. The state of CVGs is inherited by the progeny of a parasite, with stochastic switches occurring at a low frequency. Parasites with the most optimal patterns of CVGs expression are continuously selected as the environment changes, leading to adaptation and survival of the infecting population. In the first paper of this thesis, we have analyzed subcloned parasite populations to characterize, with unprecedented detail, the heterochromatin distribution associated with the active and silenced states of CVGs. This has allowed us to define different kinds of heterochromatin transitions between the active and silenced states of CVGs and has given us new insights on the regulation of var genes (one of the main virulence factors for malaria) and into the regulation of sexual conversion, a process crucial for malaria transmission. Continuing with CVG regulation, in the second paper of the thesis, we have analyzed how patterns of CVG expression are established at the onset of human infections, after passage through transmission stages. Our results suggest a loss of the epigenetic memory during transmission stages and a reset of the heterochromatin patterns that drive CVG expression. Similar patterns of CVG expression arose in different infected individuals, suggesting that the activation probability of a given CVG is an intrinsic property of the gene. In the third paper of the thesis, we have further studied the sexual conversion phenomenon. We have generated a conditional over-expression system for pfap2-g, the CVG that acts as master regulator of sexual conversion, achieving sexual conversion rates of ~90% after induction. Our results have provided new insights on how heterochromatin at different positions affects expression of pfap2-g and have allowed us to characterize the transcriptional profile of the initial stages of sexual commitment with unprecedented sensitivity. Finally, in the fourth paper of this thesis, we have studied the adaptation of the parasite to heat-shock, which happens in natural infections due to fever episodes. We expected CVGs to participate in this phenomenon, but instead we have identified pfap2-hs, a non-clonally variant transcription factor (TF), as the main driver of the heat-shock response in P. falciparum. AP2-HS acts as the functional homolog of HSF1 (a TF that drives the heat-shock response from yeast to mammals, but is absent in P. falciparum), driving a very tight transcriptional response to heat-shock, characterized by the up-regulation of hsp70 and hsp90. Although the presence of directed responses had previously been demonstrated for other cues, it is the first time that the transcription factor driving such a response is identified in P. falciparum. Taken together, the results of this thesis have broadened our knowledge of the regulation of adaptive mechanisms in P. falciparum. Learning about this deadly parasite’s defense mechanisms will be instrumental to design better strategies to fight it back in the future

Interplay of genetic, epigenetic and transcription factors in the regulation of transcriptional variation in Plasmodium falciparum

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Salut Global de Barcelona (ISGlobal)[eng] The most severe form of malaria, caused by Plasmodium falciparum parasites, still kills over half a million people every year, most of them children under the age of five. Despite huge research efforts, reduction in the global burden of disease has stalled in recent years. P. falciparum has a very complex life cycle including, among other steps, sexual reproduction in female Anopheles mosquitos and an asexual intra-erythoricitic development cycle (IDC) inside the human host, which causes the disease. During the IDC, the parasite needs to continuously adapt to changes in its environment including fluctuations in blood temperature, concentration of nutrients and other metabolites, presence of drugs, and a constant fight against the host’s immune system. In this thesis, we have studied the adaptation mechanisms of P. falciparum to this plethora of challenges, with a special focus on clonally variant genes (CVGs). In P. falciparum, CVGs are a set of genes, participating in host-parasite interactions, which can be found both in a transcriptionally active state, characterized by euchromatin, or a transcriptionally silenced state, characterized by heterochromatin. The state of CVGs is inherited by the progeny of a parasite, with stochastic switches occurring at a low frequency. Parasites with the most optimal patterns of CVGs expression are continuously selected as the environment changes, leading to adaptation and survival of the infecting population. In the first paper of this thesis, we have analyzed subcloned parasite populations to characterize, with unprecedented detail, the heterochromatin distribution associated with the active and silenced states of CVGs. This has allowed us to define different kinds of heterochromatin transitions between the active and silenced states of CVGs and has given us new insights on the regulation of var genes (one of the main virulence factors for malaria) and into the regulation of sexual conversion, a process crucial for malaria transmission. Continuing with CVG regulation, in the second paper of the thesis, we have analyzed how patterns of CVG expression are established at the onset of human infections, after passage through transmission stages. Our results suggest a loss of the epigenetic memory during transmission stages and a reset of the heterochromatin patterns that drive CVG expression. Similar patterns of CVG expression arose in different infected individuals, suggesting that the activation probability of a given CVG is an intrinsic property of the gene. In the third paper of the thesis, we have further studied the sexual conversion phenomenon. We have generated a conditional over-expression system for pfap2-g, the CVG that acts as master regulator of sexual conversion, achieving sexual conversion rates of ~90% after induction. Our results have provided new insights on how heterochromatin at different positions affects expression of pfap2-g and have allowed us to characterize the transcriptional profile of the initial stages of sexual commitment with unprecedented sensitivity. Finally, in the fourth paper of this thesis, we have studied the adaptation of the parasite to heat-shock, which happens in natural infections due to fever episodes. We expected CVGs to participate in this phenomenon, but instead we have identified pfap2-hs, a non-clonally variant transcription factor (TF), as the main driver of the heat-shock response in P. falciparum. AP2-HS acts as the functional homolog of HSF1 (a TF that drives the heat-shock response from yeast to mammals, but is absent in P. falciparum), driving a very tight transcriptional response to heat-shock, characterized by the up-regulation of hsp70 and hsp90. Although the presence of directed responses had previously been demonstrated for other cues, it is the first time that the transcription factor driving such a response is identified in P. falciparum. Taken together, the results of this thesis have broadened our knowledge of the regulation of adaptive mechanisms in P. falciparum. Learning about this deadly parasite’s defense mechanisms will be instrumental to design better strategies to fight it back in the future

Epigenetic inheritance of a phenotypically plastic epimutation

Organisms constantly have to adapt to changing environments in order to survive, thrive and successfully multiply. Phenotypic changes can be acquired by alterations of the deoxyribonucleic acid (DNA)sequence. If beneficial under natural selection, the DNA variation can become fixed permanently in a population and thereby drive its evolution. In addition to DNA sequence changes, a concept emerged that a soft, reversible layer could also potentially contribute to heritable adaptation. Epigenetic changes were shown to affect the development and complex phenotypic traits of almost isogenic organisms. Such changes can be inherited over many generations by strong self-reinforcing feedback loops without the initial trigger. Evidence for such a ‘soft’ inheritance is only just emerging and whether such phenomena are of physiological relevance in heritable adaptation though remains to be unraveled. Gene expression is regulated through several mechanisms. DNA does not exist as bare molecule, but is packaged into a highly complex structure called chromatin. Besides serving structural functions, chromatin also impacts gene expression. Chromatin can be broadly divided into transcriptionally active, gene-rich euchromatin and gene-poor, condensed heterochromatin, which serves as repressive structure for repetitive elements, such as transposons, and makes up most of the euchromatic genome. In some organisms, nuclear small ribonucleic acid (RNA) pathways are essential to initiate and maintain constitutive heterochromatin. The centerpiece of such pathways is a small RNA-bound Argonaute protein, which binds by complementary base-pairing to nascent transcripts and subsequently recruits effector complexes that mediate silencing. Given the appropriate small RNA, this pathway can theoretically target any expressed locus, thereby making it a versatile silencing strategy. In nematodes, small RNAs were shown to induce stable silencing of some protein coding genes that can be epigenetically maintained over tens of generations. During my PhD, I studied RNA interference (RNAi)-mediated epigenetic phenomena in the fission yeast Schizosaccharomyces pombe (S. pombe). In S. pombe, RNAi-mediated silencing is under strong negative control and can only be initiated in the presence of an enabling mutation, such as in genes encoding subunits of the RNA polymerase-associated factor 1 complex (Paf1C). On one hand, such mutations can have a detrimental effect on viability. On the other hand, the silencing phenotype observed in Paf1C mutants cannot be inherited to wild-type cells, suggesting that also all marks of the silencing event were erased. If RNAi-mediated epigenetic phenomena also exist in wild-type cells was not known. My main achievement during PhD was to discover that wild-type S. pombe cells remember a parental silencing event through acquiring a phenotypically neutral epimutation. I could show that such epimutation does not cause gene silencing when inherited by wild type cells. Yet, upon repeated mutation of Paf1C, the silencing phenotype was reinstated in subsequent generations. I could further show that the phenotypically neutral epimutation entails high levels of small interfering RNA (siRNA) and histone 3 lysine 9 tri-methylation (H3K9me3), and that its transgenerational inheritance depends on RNAi and H3K9 methylation. This finding is astounding, because H3K9me3 has commonly been associated with gene repression. That we have not observed silencing, despite high enrichments of this mark, was therefore highly unexpected. Based on my findings, I conclude that H3K9me3 is not repressive per se, but rather functions as stable epigenetic mark that can retain information of a previous gene-silencing event. Upon deposition of H3K9me3, the silencing phenotype is dependent on the modulation of Paf1C function. The discovery of this distinct form of epigenetic memory lets me speculate that it may have evolved to allow population adaptation to dynamic environments

Aerospace medicine and biology: A continuing bibliography with indexes, supplement 118

This special bibliography lists 338 reports, articles, and other documents introduced into the NASA scientific and technical information system in July 1973

Chromosomes in Interphase and Mitosis

The three-dimensional organization of the chromatin fiber is driven by entropy. Therefore, the folding of the chromatin fiber is essentially a problem of statistical physics. In the present thesis, two questions in the context of chromatin folding which are still not fully understood are investigated: on the one hand the organization of chromatin in mitosis and on the other hand the changes of chromatin organization in the damage response to ionizing radiation. In the first part we develop a model that explains the condensation of mitotic chromosomes by size-restricted dynamic looping of the chromatin fiber. Our results show also that chromatin loops can contribute to the experimentally determined bending rigidity of mitotic chromatids and generate the correct force-extension behaviour. In a next step, this folding model is then extended to describe sister chromatids by including dynamic binding and unbinding of sister fibers. We assess the interplay between cohesion and condensation and show that alignment and cohesion of sister chromatids requires detailed control of the abundance of tethering points between them. In the second part we examine the damage response of interphase chromosomes. With an expression-dependent folding model and utilizing experimental data on the transcriptional activity of cells that were exposed to ionizing radiation, we first show that the overall organization of chromatin does not change after irradiation. By modeling actual fiber breaks in local environments we demonstrate that broken ends are passively transported to the surface of their domains and that this facilitates recognition of the break by diffusing proteins. Finally, we use a graph theoretical approach to analyze the structural changes of histone positions in localization microscopy images of cells that were exposed to ionizing radiation. We validate our previous results that no changes of the overall organization of chromatin is recognizable and demonstrate that highly packaged heterochromatic areas of the genome decondense upon irradiation