DeCRyPT, Deciphering Cis-Regulatory Principles of Transcriptional regulation, aimed to functionally dissect cis-regulatory landscapes and uncover general principles of genome regulation during embryonic development by combining population genetics, developmental genetics, and novel genomic methods using Drosophila as a model.
Aim 1 used population genetics to uncover new mechanisms of gene regulation. Using F1 embryos from 8 genotypes at 3 embryonic stages, we determined the impact of genetic diversity across different regulatory layers. Th comprehensive dataset enabled using combined haplotype testing and AI-based deep neural networks. This uncovered several surprises: (1) Genetic variation impacts gene expression more frequently than chromatin features. (2) Allelic imbalance in regulatory elements during embryogenesis is common and highly heritable. (3) Variation in RNA is more predictive of variation in H3K4me3 than vice versa. (4) The ability to buffer genetic variants is influenced by regulatory complexity. (5) The model revealed new partners for well-studied transcription factors, e.g. CTCF. (6) We solved the challenge of obtaining cell type specific effects of sequence variation. Aim 1 resulted in 3 publications (PMC7849415, PMC8734213, Sigalova et al. doi: 10.1101/2024.10.24.619975) with a 4th in preparation.
Aim 2 dissected functional regulatory domains through genomic deletions of regulatory elements in embryos, measuring their impact on genome topology and expression to assess properties like long-range regulation, enhancer (E) sharing and redundancy. (1) This systematic assessment of E deletion revealed extensive redundancy under normal conditions, but when the embryos are placed under environmental stress, these Es become essential (Dulja, in prep). (2) To identify examples of long-range or shared enhancers we performed high-resolution captureC to link enhancers to their potential target genes and identify potential long-range E-P pairs for deletion. This revealed an interesting switch in E-P communication during embryogenesis. (3) Functionally dissecting long-range chromatin loops made an unexpected discovery. Many are gene-gene loops, bringing 2 genes with related function together. Through deletions we showed that the loop is essential to coordinate their relative levels of co-expression. Aim 2 resulted in 2 publications (PMC11018526, PMID: 38157845) and 1 in preparation (Dulja et al.).
Aim 3 altered the regulatory context in which enhancers function by reshaping TADs and deciphered the pairing rules of TAD boundaries in Drosophila. (1) To determine when and how TADs are formed, we generated loss-of-function embryos (both maternal and zygotic) for CTCF, BEAF-32 and CP190, and determined their requirement for TAD structure and gene expression. Removing any one insulator had little global impact on the establishment of TADs, although some boundaries (10%) had reduced insulation. Our results suggest context-dependent redundancy and new insulator proteins. (2) To change the size and content of a gene’s regulatory domain, we used large-scale inversions and deletions which resulted in TAD fusion and mixing. This changed the context of the gene’s regulatory domain yet had surprisingly little effect on gene expression in most cases, although it does for some, opening many new research directions. (3) By performing large scale boundary insertions, we identified genomic elements that can form a new boundary in an ectopic location, and those that can’t. Known insulator binding is not predictive of working boundaries. Interestingly, many working boundaries are orientation and/or context-dependent. We identified new motif pairs highly predictive of working boundaries, including motifs for unknown factors and has important evolutionary implications for chromatin organisation (Varisco, Cavalheiro, in prep). Aim 3 resulted in 2 publications (PMC9897672, PMC7116017), and 1 in preparation (Varisco, Cavalheiro et al.).
