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3D Organization of Functionally Conserved Cis-Regulatory Elements

Periodic Reporting for period 1 - 3Dcis (3D Organization of Functionally Conserved Cis-Regulatory Elements)

Período documentado: 2018-04-01 hasta 2020-03-31

Precise gene expression during embryonic development is crucial to generate the numerous cell types and tissues that build the animal body plan. This patterning is driven by developmental genes that are often expressed in a complex and conserved fashion. Gene expression patterns are instructed by regulatory sequences in the genome, the so-called enhancers. Enhancers act as switches in the genome that can modulate gene expression levels and turn a gene on or off. Given this important function for gene regulation, it is surprising that enhancers are often not conserved at the sequence level and act rather as seemingly variable entities during genome evolution. However, the mechanisms that allow this regulatory plasticity during evolution, but also maintenance of conserved genes expression during development are not well understood. Enhancers together with their target genes are spatially organized into conserved 3D chromatin structures, suggesting that spatial conformation within the nucleus is the missing link between less conserved enhancers and conserved target gene expression during animal development.
This project aims to understand the functional relationship between enhancers, conserved gene expression patterns and 3D chromatin structure during vertebrate development and evolution. As sequence conservation of enhancers is extremely limited, we applied innovative approaches to identify and systematically compare the functional response of enhancers and gene expression across different animal lineages. We identified functionally conserved enhancers and their target genes in the distantly related vertebrate model organisms, zebrafish (Danio rerio) and the western clawed frog (Xenopus tropicalis). Assessing the underlying 3D chromatin architecture, we deciphered the chromatin structures at orthologous genomic regions and enriched our understanding of how the cis-regulatory information of enhancers and genome structure translate into function. Although enhancers are not constrained by sequence conservation, they are constrained by conserved structural properties in the nucleus. Our data pave the way towards a deeper understanding of cis-regulatory mechanisms during development and evolution but also towards a better evaluation of regulatory mutations that are frequently detected in human congenital disease and cancer.
A few highly conserved signalling pathways are active during animal body plan formation. Signalling pathways, such as fibroblast growth factor (FGF) and retinoic acid (RA) control the expression of effector genes by regulating specific DNA-binding proteins that bind at enhancers. Using biologically active molecules, we impaired or stimulated several signaling cascades during germ-layer formation in Xenopus and Zebrafish. We profiled open chromatin regions with ATAC-sequencing to identify a differential response in enhancers and measured changes in gene expression by RNA-sequencing. Although 400 million years of evolution separate the two species, comparing their functional response we identified a basic regulatory network of enhancers and target genes controlled by these pathways. We then measured chromatin interactions and 3D structure in both species during development, using various chromosome conformation capture assay derivatives. Determining the relative position of enhancers to the here identified 3D structure of chromatin extended our previous knowledge on the evolution of cis-regulatory information. The differences and similarities observed in the number and position of enhancers correlated well with the associated 3D chromatin structure. Furthermore, several features of chromatin structure were highly predictive for a stable number and position of enhancers. Together, the results of this research project revealed cis-regulatory mechanisms driving conserved gene expression patterns and common body structures, but also the constraints that different animal lineages have faced during their evolutionary path.
Less than 5% of our genome encodes protein-coding genes. The rest, more than 95%, is non-coding DNA that harbors, among others, regulatory elements such as enhancers and elements mediating the 3D genome structure. Whole-genome sequencing approaches in diagnostic settings detect an ever-increasing number of non-coding variances and regulatory mutations. A deeper understanding of how the non-coding DNA functions in development and evolution to regulate genes will account for major challenges in human genetics. This project combines several aspects from basic research fields, including Gene Regulation, 3D Genome Architecture, Evolution and Developmental Biology. However, the implementation of the research results from this project into diagnostic settings will contribute to better predict and distinguish neutral sequence variations from disease-causing mutations.