Background Low-frequency variants play an important role in breast cancer (BC) susceptibility. Gene-based methods can increase power by combining multiple variants in the same gene and help... Show moreBackground Low-frequency variants play an important role in breast cancer (BC) susceptibility. Gene-based methods can increase power by combining multiple variants in the same gene and help identify target genes.Methods We evaluated the potential of gene-based aggregation in the Breast Cancer Association Consortium cohorts including 83,471 cases and 59,199 controls. Low-frequency variants were aggregated for individual genes' coding and regulatory regions. Association results in European ancestry samples were compared to single-marker association results in the same cohort. Gene-based associations were also combined in meta-analysis across individuals with European, Asian, African, and Latin American and Hispanic ancestry.Results In European ancestry samples, 14 genes were significantly associated (q < 0.05) with BC. Of those, two genes, FMNL3 (P = 6.11 x 10(-6)) and AC058822.1 (P = 1.47 x 10(-4)), represent new associations. High FMNL3 expression has previously been linked to poor prognosis in several other cancers. Meta-analysis of samples with diverse ancestry discovered further associations including established candidate genes ESR1 and CBLB. Furthermore, literature review and database query found further support for a biologically plausible link with cancer for genes CBLB, FMNL3, FGFR2, LSP1, MAP3K1, and SRGAP2C.Conclusions Using extended gene-based aggregation tests including coding and regulatory variation, we report identification of plausible target genes for previously identified single-marker associations with BC as well as the discovery of novel genes implicated in BC development. Including multi ancestral cohorts in this study enabled the identification of otherwise missed disease associations as ESR1 (P = 1.31 x 10(-5)), demonstrating the importance of diversifying study cohorts. Show less
Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing... Show moreGrainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER?/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER?/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Show less
The human body consists of hundreds, perhaps thousands of different types of cells, each with different morphologies and functions, despite having the same genome. This diversity is created by gene... Show moreThe human body consists of hundreds, perhaps thousands of different types of cells, each with different morphologies and functions, despite having the same genome. This diversity is created by gene regulation, a set of mechanisms that determine, which genes are used to make proteins and which genes are kept silent. During embryonic development, gene are turned on and off in a tightly orchestrated manner, to make sure that the right cell type is created at the right time and place.In this thesis we report several studies pertaining to gene regulation in embryonic development. Each of the four chapters will cover a different layer of the gene regulation toolbox: gene inactivation by DNA methylation, transcriptional regulation in the developing kidney, regulation of protein turnover and translational regulation through micro-RNAs. Together, these studies provide a refined understanding of the crucial role of gene regulation for embryonic development. Show less
BackgroundFrequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and... Show moreBackgroundFrequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions.ResultsWe first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on Enhancer(TRAM2), as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness.ConclusionsYAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis. Show less
Aspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the... Show moreAspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the complex regulation of the expression of pectinolytic genes in A. niger. Chapter 1 gives an introduction of filamentous fungi with a focus on A. niger, and their industrial application. Chapter 2 describes a split marker approach in combination with NHEJ mutants for efficient targeted gene deletion. In Chapter 3, we constructed a set of isogenic auxotrophic strains by recycling pyrG marker. In Chapter 4, we selected five galacturonic acid (GA) induced genes to analyze the regulation of promoter activity of these genes in vivo by constructing promoter_amdS reporter strains. In Chapter 5, we identified the GA responsive transcription activator GaaR of A. niger by homology to BcGaaR of Botrytis cinerea. In Chapter 6 full genome sequencing of five mutants showing constitutive pectinolytic activity revealed allelic mutations in one particular gene gaaX. GaaX is likely to act as a transcription repressor which inhibits GaaR activity under non-inducing conditions. Chapter 7 summarizes and discusses the major conclusions of this thesis and proposes some future directions to study the regulatory mechanism of pectin degradation in A. niger. Show less
The work described in this thesis focuses on the mechanisms that give rise to alternative mRNAs and their alternative translation into proteins. Each of the described studies has been based on a... Show moreThe work described in this thesis focuses on the mechanisms that give rise to alternative mRNAs and their alternative translation into proteins. Each of the described studies has been based on a specific set of high-throughput RNA sequencing technologies. An overview of the available RNA sequencing methods, together with an introduction to different regulatory layers which define the expression of a gene, are presented in Chapter 1. Our work in Chapter 2 and Chapter 3 investigates the process of alternative polyadenylation. Chapter 2 shows the role of alternative polyadenylation in the context of oculopharyngeal muscular dystrophy. Chapter 3 describes genetic variants associated with alternative polyadenylation. Chapter 4 focuses on mechanisms controlling protein synthesis (translation) during skeletal muscle differentiation, highlighting changes in the use of alternative translation initiation sites. In Chapter 5 we investigated the interdependence between alternative regulatory events in gene expression. In this study, based on single-molecule full-length RNA sequencing, we demonstrated coordination and interdependence between alternative transcription initiation, alternative splicing, and alternative polyadenylation. Finally, Chapter 6 connects fundamental research in the RNA field with clinical care, describing new diagnostic and therapeutic approaches. Show less
Temporal variation in abiotic and biotic variables such as temperature, rainfall, food availability or predation pressure profoundly affects the abilities of organisms to survive and reproduce... Show moreTemporal variation in abiotic and biotic variables such as temperature, rainfall, food availability or predation pressure profoundly affects the abilities of organisms to survive and reproduce successfully. Most organisms are remarkably flexible in the face of such heterogeneity in habitat quality, and display phenotypic plasticity in response to environmental variation, i.e. the production of alternative phenotypes from a single genotype, dependent on the experienced environment. The aftrotropical butterfly Bicyclus anynana expresses alternative adult life histories in its habitat's wet and dry seasons, including reproductive timing and lifespan. This thesis aims to increase insight into the hormonal and transcriptional patterns that underlie life history plasticity in B. anynana. The first question is how the environment experienced during development induces the two adult seasonal forms via conserved hormonal pathways. The second major question covered in this thesis is what transcriptional changes in the adult are associated with the seasonal forms, and how ageing differs between the seasons. Together, these data contribute to a better mechanistic understanding of plastic responses as adaptation to environmental variation, and provide starting points for research into mechanisms linking development and ageing in humans, and how events during early development can affect lifespan and human health. Show less
Gene expression is a complicated process with multiple types of regulation, including binding of proteins termed transcription factors. This thesis looks at transcription factors and transcription... Show moreGene expression is a complicated process with multiple types of regulation, including binding of proteins termed transcription factors. This thesis looks at transcription factors and transcription factor binding site discovery through computational predictions and wet lab work to better elucidate their role in transcriptional regulation. This includes bioinformatics tools to extrapolate transcription factors common to a set of co-regulated sequences, such as genes differentially expressed in a microarray or next-generation sequencing experiment. It also includes a working pipeline to analyze next-generation sequencing data, used in the following projects: Next-generation sequencing of chromatin-immunoprecipitated CBP and p300 (two highly homologous transcription factors) bound DNA was performed to analyze their (different) roles in cell cycle regulation. Next-generation sequencing of RNA from differentiating muscle cells was also done to identify differential gene expression during myogenesis, as well as identify novel promoter regions (a common target of transcription factors). Taken together, computational and wet lab tools can enhance our knowledge of transcriptional regulation, as described by several applications to enhance our knowledge of myogenic and cell-cycle regulation in this thesis. Show less
