PROJECT ABSTRACT A fundamental question in regulatory biology is whether enhancers regulate only the coding target genes with spatial proximity, or whether they regulate chromosomal architecture, exerting transcriptional effects on genes far removed in a specific chromosome. Thus, in addition to exhibiting regulated interactions with its cognate promoter, and perhaps with other enhancers in a single TAD, it is important to solve whether signal-induced proximity of specific robustly-activated regulatory enhancers, separated by vast linear distances within a chromosome, constitute a “first tier” network that alters the transcription of specific, interacting component enhancers. We hypothesize that this ”first tier” network, while not impacting the ability of each component enhancer to loop to and activate its cognate target coding gene promoter, nucleates formation of an architectural “structure” that provides the machinery that licenses the robustness of the transcriptional response imparted by these individual enhancers. We will use both GRO-seq and assays of 3D architecture to test whether conceptualize a ligand-dependent distributive superenhancer connectome dictates chromosomal architecture. This would represent an entirely new perspective on enhancer networks, revealing interactions of E2-regulated enhancers that modulate whole chromosome architecture and ensemble chromosomal structures result in an unexpected integrated transcriptional response network based on actions of single, robust “first tier” enhancers.