A multimodal MPRA platform to study regulatory sequence function

SUMMARY The overarching goal of the proposed research is to develop tools capable of elucidating the mechanisms and grammar of how the regulatory elements in the genome encode the interactions between chromatin, regulatory factors, and transcriptional processes that underlie the precise regulation of transcription. To study the mechanisms of how individual regulatory elements regulate different aspects of transcription, we propose to develop technology based on massively parallel reporter barcoding strategies to allow interrogation of the different molecular aspects of regulatory element function, either in isolation or combination, while separated from the variegating influence of their genomic sequence environment. The new assays will enable simultaneous measurements of the effect of sequence perturbations on chromatin state, DNA-protein interactions, 3D chromatin interactions, and promoter-proximal aspects of transcription regulation (initiation, pausing, pause release). The new methodology will be validated against the effects of introducing corresponding mutations at endogenous genomic loci by genome editing. To demonstrate the use of these novel assays that will simultaneously provide orthogonal data on the functional characteristics of thousands of enhancer perturbations, we will test the hypothesis that individual enhancers may exert their function through modulation of different transcriptional mechanisms. This will include testing whether individual enhancers affect transcription initiation or promoter pause release, and whether enhancers function during cellular differentiation or acute stimulus responses. To this end, we will establish a process to integrate the MPRA reporters into defined loci in human iPS cells, and develop software to facilitate reporter sequence design, data analysis and integration with genome-wide profiling data.