Identification of cell-type-specific multi-way regulatory hubs during vertebrate development

• Applicant: Scott Lacadie, Ph.D.
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology ; Bioinformatics and Theoretical Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 439822597

Regulation of gene expression is the driving force behind animal diversity, not the number and sequence of proteins. Cis regulatory elements (CREs) are DNA sequences that determine when and where genes are expressed. Their activity is thought to determine cell-type specification during development, cellular response to the environment, and to be altered in many human disease states. Several minimal CREs, spanning tens of kilobases in linear genome distance, have been shown to act on a given gene in a single cell type. Out of this observation was born the hypothesis of the CRE “hub”, where several CREs form a conglomerate of three-dimensional (3D) interactions which acts as a single unit to drive gene expression. CRE hubs remain controversial due to difficulties in measuring multi-way interactions directly. The first objective of our proposal is to use single-nucleus ATAC-seq (sci-ATAC-seq) to predict CRE hubs as local groups of highly co-accessible CREs and to verify their existence during zebrafish embryonic development with our novel assay for multi-way 3D interactions, NPC-walks.Sci-ATAC-seq data has allowed us to characterize the regulatory code behind cell-type specificity in the whole prim-5 stage zebrafish embryo. Though such single-cell resolution assays can predict cell-state sub-populations from heterogeneous samples with significant confidence, it is difficult and rare to verify such predictions with experiments. To this end, in Objective 2 we will measure accessibility and gene expression at single-cell resolution in zebrafish embryos carrying the cloche allele. Cloche mutant embryos have been previously shown to have severely impaired differentiation of endothelial and hematopoietic cell types, therefore the cell-type accessibility and expression maps should be substantially altered in mutant animals. Furthermore, we will profile cloche embryos for multi-way CRE 3D interactions using NPC-walks to confirm predictions of endothelial and hematopoietic-specific CRE hubs and their targets from Objective 1.The results of the proposed projects will immediately provide a key resource of tissue-specific CRE interactions and will address several current and pressing hypotheses in the field regarding the existence of 3D CRE hubs and their tissue specificity during development. They will build a foundation for future studies that will relate 3D genome architecture with all aspects of gene regulation, leading to a holistic and super-resolved understanding of vertebrate development at both the cellular and molecular levels.

• DFG Programme: Research Grants