Human pluripotent cells hold great promise for numerous biomedical applications including eventual use in regenerative medicine and personalized therapies. However, achieving this important goal requires a better understanding of the regulatory circuitry that establishes and maintains molecular and functional pluripotency. Our previous proposal has provided many profound new insights, data sets and tools for the scientific community. We characterized transcription factor and epigenome dynamics, uncovered hundreds of new non-coding DNA and long non-coding RNA (lncRNA) elements and began to assemble a comprehensive, multi-layered pluripotent gene regulatory network. Through advancements in technology, experimental systems and computational analysis we are now uniquely poised to take the next leap forward in “Dissecting the establishment and regulation of human pluripotency”. Our current objective is to leverage these latest biological, experimental and computational advances to close important gaps and provide a comprehensive, functional characterization of the regulatory landscape and hierarchies in pluripotent stem cells as well as their dynamics during reprogramming. Specifically we will focus on the following pressing questions that have arisen over the past years: 1) What is the exact nature of the pluripotent state(s) (Projects 1-3)? We already a comprehensive catalogue of the regulatory elements and defining features to will help distinguish these states and will complete these at unprecedented resolution. Why does the naïve state emerge only transiently during human reprogramming (Project 1 and 3)? What are the implications of deregulating the pluripotent network including its cis regulatory elements (Project 3)? What is the contribution of genetic variation and coding/non-coding transcription (Project 2 and 3)? 2) How is the pluripotent genome organized in three-dimensional space to establish pluripotency gene- expression programs (Project 1-3)? It is becoming clear that lncRNAs can bind and influence the organization of chromatin within the nucleus. We aim to understand molecular underpinnings of how RNA (Project 2), DNA (Project 3) and epigenetic/transcription factors (Project 1) establish the nuclear architecture of the pluripotent genome. We will develop novel visualization and genomic technologies in living and fixed cells to chart the dynamic of reorganizing these factors (Project 2) and their functional implications in the process of reprograming as well as during the maintenance of pluripotency (Project 1, 2 and 3). 3) What are the cis regulatory elements that reinforce the pluripotent state (Project 3)? In our previous proposal we have identified numerous DNA cis regulatory modules (CRMs) and began to unravel RNA CRMs that guide nuclear localization. We now aim to functionally characterize the sequences and motifs within CRMs on a genome-wide scale (Project 2 and 3).