Tight cooperation between Mot1p and NC2β in regulating genome-wide transcription, repression of transcription following heat shock induction and genetic interaction with SAGA

TATA-binding protein (TBP) is central to the regulation of eukaryotic transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1p or the NC2 complex. Recent evidence suggests that Mot1p, NC2 and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1p and NC2βduring basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes. Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2β depletion during heat shock resulted in failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2β displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results support the model that Mot1p and NC2β directly cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress

Regulatory DNA and Stem Cells in Complex Genetic Diseases

Delineating the pathogenesis of complex genetic diseases is complicated by the great variety of genetic loci, genes, cell types, environmental factors and tissues that are involved. The genetic background of complex genetic diseases has been intensively studied through genome wide association studies (GWASs). This led to the association of many genetic loci to a multitude of diseases. The identification of causal variants and affected genes has proven difficult, thereby leaving some pathogenic mechanismsunresolved and potential therapeutic targets unrevealed. In this thesis, we study the involvement of DNA regulatory elements (DRE) in the pathogeneses of complex genetic diseases and epithelial mechanisms that are potentially involved in inflammatory bowel disease (IBD). In the first part of this thesis we studied loci associated to IBD, CVD cardiovascular diseases) and CKD (chronic kidney disease). We show that disease associated loci are enriched for active DRE and we used chromatin conformation capture to study the 3D configuration of these loci. Through this approach we identified many novel candidate genes, pathways and key regulators of IBD, CVD and CKD.Genetic studies on IBD have shown that the intestinal epithelium plays a important role in the pathogenesis of this complex genetic disease. Therefore, in the second part of this thesis, we use human intestinal organoids to study the interaction between bacterial antigens and the intestinal epithelium. We show that intestinal stem cells are a major source of the inflammatory response and that the responsiveness of the epithelium is regulated at the post-transcriptional level

Regulatory DNA and Stem Cells in Complex Genetic Diseases

Delineating the pathogenesis of complex genetic diseases is complicated by the great variety of genetic loci, genes, cell types, environmental factors and tissues that are involved. The genetic background of complex genetic diseases has been intensively studied through genome wide association studies (GWASs). This led to the association of many genetic loci to a multitude of diseases. The identification of causal variants and affected genes has proven difficult, thereby leaving some pathogenic mechanismsunresolved and potential therapeutic targets unrevealed. In this thesis, we study the involvement of DNA regulatory elements (DRE) in the pathogeneses of complex genetic diseases and epithelial mechanisms that are potentially involved in inflammatory bowel disease (IBD). In the first part of this thesis we studied loci associated to IBD, CVD cardiovascular diseases) and CKD (chronic kidney disease). We show that disease associated loci are enriched for active DRE and we used chromatin conformation capture to study the 3D configuration of these loci. Through this approach we identified many novel candidate genes, pathways and key regulators of IBD, CVD and CKD.Genetic studies on IBD have shown that the intestinal epithelium plays a important role in the pathogenesis of this complex genetic disease. Therefore, in the second part of this thesis, we use human intestinal organoids to study the interaction between bacterial antigens and the intestinal epithelium. We show that intestinal stem cells are a major source of the inflammatory response and that the responsiveness of the epithelium is regulated at the post-transcriptional level

Non-coding DNA in IBD : From sequence variation in DNA regulatory elements to novel therapeutic potential

Genome-wide association studies have identified over 200 loci associated with IBD. We and others have recently shown that, in addition to variants in protein-coding genes, the majority of the associated loci are related to DNA regulatory elements (DREs). These findings add a dimension to the already complex genetic background of IBD. In this review we summarise the existing evidence on the role of DREs in IBD. We discuss how epigenetic research can be used in candidate gene approaches that take non-coding variants into account and can help to pinpoint the essential pathways and cell types in the pathogenesis of IBD. Despite the increased level of genetic complexity, these findings can contribute to novel therapeutic options that target transcription factor binding and enhancer activity. Finally, we summarise the future directions and challenges of this emerging field

Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium

BACKGROUND & AIMS: The contribution of genetic factors to the pathogenesis of inflammatory bowel disease (IBD) has been established by twin, targeted-sequencing, and genome-wide association studies. These studies identified many risk loci, and research is underway to identify causal variants. These studies have focused mainly on protein-coding genes. We investigated other functional elements in the human genome, such as regulatory regions. METHODS: Using acetylated histone 3 lysine 27 chromatin immunoprecipitation and sequencing, we identified tens of thousands of potential regulatory regions that are active in intestinal epithelium (primary intestinal crypts and cultured organoids) isolated from resected material and from biopsies collected during ileo-colonoscopies and immune cells (monocytes, macrophages, CD34(+), CD4(+), and CD8(+)). We correlated these regions with susceptibility loci for IBD. RESULTS: We have generated acetylated histone 3 lysine 27 profiles from primary intestinal epithelium and cultured organoids, which we have made publically available. We found that 45 of 163 single nucleotide polymorphisms (SNPs) associated with IBD overlap specifically with active regulatory elements. In addition, by taking strong linkage disequilibrium into account, another 47 IBD-associated SNPs colocalized with active regulatory elements through other SNPs in their vicinity. Altogether, 92 of 163 IBD-associated SNPs correlated with distinct active regulatory elements-a frequency 2.5- to 3.5-fold greater than that expected from random sampling. The variations in these SNPs often create or disrupt known binding motifs; they might affect the binding of transcriptional regulators to alter expression of regulated genes. CONCLUSIONS: In addition to variants in protein coding genes, variants in noncoding DNA regulatory regions that are active in intestinal epithelium and immune cells are potentially involved in the pathogenesis of IBD

Chromatin Conformation Links Distal Target Genes to CKD Loci

Genome-wide association studies (GWASs) have identified many genetic risk factors for CKD. However, linking common variants to genes that are causal for CKD etiology remains challenging. By adapting self-transcribing active regulatory region sequencing, we evaluated the effect of genetic variation on DNA regulatory elements (DREs). Variants in linkage with the CKD-associated single-nucleotide polymorphism rs11959928 were shown to affect DRE function, illustrating that genes regulated by DREs colocalizing with CKD-associated variation can be dysregulated and therefore, considered as CKD candidate genes. To identify target genes of these DREs, we used circular chromosome conformation capture (4C) sequencing on glomerular endothelial cells and renal tubular epithelial cells. Our 4C analyses revealed interactions of CKD-associated susceptibility regions with the transcriptional start sites of 304 target genes. Overlap with multiple databases confirmed that many of these target genes are involved in kidney homeostasis. Expression quantitative trait loci analysis revealed that mRNA levels of many target genes are genotype dependent. Pathway analyses showed that target genes were enriched in processes crucial for renal function, identifying dysregulated geranylgeranyl diphosphate biosynthesis as a potential disease mechanism. Overall, our data annotated multiple genes to previously reported CKD-associated single-nucleotide polymorphisms and provided evidence for interaction between these loci and target genes. This pipeline provides a novel technique for hypothesis generation and complements classic GWAS interpretation. Future studies are required to specify the implications of our dataset and further reveal the complex roles that common variants have in complex diseases, such as CKD

Additional file 5: Table S6. of Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease

Upstream regulators. (XLSX 135 kb

Additional file 7: Table S3. of Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease

Publicly available datasets. (XLSX 11 kb