PROJECT SUMMARY / ABSTRACT A fundamental challenge in developmental biology is to dissect how one multipotent cell differentiates into a specific cell type. Most studies are limited to 1-dimensional genomic data that measure transcription level (RNA- seq), protein binding intensity (ChIP-seq), and chromatin accessibility (ATAC-seq). These datasets lack direct evidence of communication between various regulatory elements that accommodate gene regulation and differentiation. To solve this problem, we will leverage cutting-edge 3D genome technologies, ChIA-PET and ChIA-Drop. By enriching for specific protein factors CCCTC binding factor (CTCF) and RNA Polymerase II (RNAPII), one can interrogate chromatin architecture and gene regulation in aggregated bulk cells (ChIA-PET) and in a single molecule (ChIA-Drop). We will exploit the highly dissimilar genomes in F1 hybrid mouse strains derived from mating a laboratory mouse and a wild mouse to assign high-throughput sequencing reads to parental origin, thereby unraveling the allele-specific gene expression and chromatin interactions. We propose to: (i) determine whether allele-specific interactions between regulatory elements and methylation status in mouse embryonic stem cells (mESCs) drive allele-specific gene expression, (ii) quantify cell-to-cell heterogeneity of multiplex chromatin interactions. We will subsequently differentiate mESCs into three lineage-specific precursors ectoderm, mesoderm, and endoderm in vitro. By performing ChIA-PET, we can identify which, if any, of the pre-established interactions among enhancers, promoters, and CTCF persist or vanish after this process. ChIA-Drop data will potentially capture the dynamics therein. Throughout the K99 and R00 phases, we will continue to develop computational algorithms that can: (i) quantitatively assess reproducibility of replicate experiments, (ii) identify statistically significant differential interactions, and (iii) trace and quantify single- molecule dynamics and heterogeneity of allele-specific multiplex interactions. To succeed in these aims, the investigator will expand her knowledge domain to developmental biology and receive additional hands-on experimental training in 3D genome mapping technologies and mouse embryonic stem cell culture, harvest, and differentiation techniques. Together, these genome-wide communication links between regulatory elements and architectural protein will provide insights into gene regulation and genomic imprinting mechanisms during gastrulation.