The goal of this proposal is to determine whether gene regulation is the cause or consequence of three- dimensional (3-D) genome organization. Enhancers are cis-regulatory elements that drive spatiotemporal gene expression from their target promoter. Disruption of enhancer-promoter (E-P) interactions can result in severe developmental disorders and congenital malformations. Enhancers typically communicate with their cognate promoter within 3-D features of genome folding called topologically associating domains (TADs). These features were originally characterized by proximity ligation sequencing techniques (ie: Hi-C). The depletion of two architectural proteins, either CTCF or cohesin, resulted in the dissolution of TADs by Hi-C; however, imaging-based approaches revealed that 3-D structures remained. Moreover, the effect of architectural protein depletion on gene expression was relatively mild, suggesting that E- P communication is robust to TAD dissemination. These perplexing findings have left the field of genome organization divided about the formation and function of TADs. One hypothesis posits that E-P interactions give rise to TAD structure. The other hypothesis is that architectural proteins form TADs in order to facilitate the E-P interactions within. I hypothesize that both gene regulatory elements and architectural proteins contribute to 3-D topology. I will test the contribution of each model in a unified system and defined developmental context. In order to retain in vivo spatiotemporal information at single-cell resolution, I will investigate the 3-D organization of the Sonic hedgehog (Shh) TAD in mouse embryonic brain tissue using a fluorescence in situ hybridization (FISH) approach. I designed small (10 kilobase) DNA-FISH probes to measure the physical distances between E-P elements. My preliminary data for one E-P pair, revealed both enhancer-dependent and enhancer-independent proximity in specific regions of the developing brain. While the enhancer-dependent proximity supports the model of active enhancers in mediating 3-D structure, I hypothesize that the enhancer- independent proximity is mediated by architectural proteins.
In Aim 1, I will map all E-P interactions for the Shh locus using sequential DNA-FISH. I will then determine the contribution of enhancers and architectural proteins to the locus? configuration by using mutants devoid of enhancers and specific CTCF binding sites, respectively. The experiments in Aim 2 will explore complex E-P communication of two redundant Shh enhancers. I will analyze the transcriptional output and spatial organization of the redundant enhancers and determine if these metrics are altered in the absence of the reciprocal enhancer. Taken together, the data from this proposal will create a paradigm for understanding how combinatorial gene regulation intersects with 3-D genome organization. Importantly, preserving the in vivo developmental context will be invaluable for translating how E-P miscommunication results in developmental disorders and disease.
Mammalian development requires enhancer-promoter (E-P) communication for spatiotemporal gene expression and cell fate determination during embryogenesis. E-P interactions coincide with three-dimensional (3-D) topological features, however, it is currently unclear if E-P interactions are the cause or consequence of 3-D features. Investigating E-P communication in 3-D space will provide fundamental knowledge of gene regulation, 3-D genome organization, and how miscommunication among these elements contributes to human developmental disease, including congenital malformations.
