One of the biggest surprises of the human genome project was the vast amount of transcription observed in the noncoding genome, comprised of thousands of long noncoding RNAs (lncRNA). In previous work, we identified a few lncRNAs that contribute important functional roles in human reprograming and maintenance of pluripotency. Our previous proposal, in collaboration with the Drs. Meissner's and Gnirke's proposals, has now identified more than a hundred lncRNAs that are specific to the pluripotent state, dynamically regulated during reprograming and enriched to reside near key developmental regulators. We have also identified an emerging mechanistic theme of lncRNAs as regulatory factors in shaping nuclear architecture and, in turn, driving pluripotent gene expression programs. Based on these studies we now propose to take the next leap forward towards addressing the following pressing questions: Aim 1: Do pluripotent specific expressed lncRNA loci that are in proximity to pluripotency and developmental regulators functionally contribute to the maintenance of pluripotency? It is tempting to think that lncRNAs that reside near key pluripotency and developmental regulators may function in cis to either activate pluripotency factors or repress differentiation genes. Here we will systematically apply a transcriptional inhibition (CRISPR-i) screen on a hundred of these lncRNA loci for those that are required to maintain pluripotency. We will further perform numerous individual validation tests and RNase-H mediated depletion of lncRNAs in parallel. Finally, we will determine influences on pluripotent and differentiation gene-expression programs upon lncRNA loss-of function (LOF) using massively-parallel RNA-sequencing technologies. Aim 2: What are the genome wide RNA-DNA and RNA-Protein localization properties of nuclear lncRNAs that facilitate proper pluripotency gene-expression programs? We have revised the proposal for a deeper focus on FIRRE and CISTR-ACT, two lncRNAs that, as determined in the previous proposal, share key properties highly relevant to the proposed study: required for stemness, shared mechanism of facilitating nuclear architecture and strongly linked to human disease. By performing loss of function studies on identified protein partners we can begin to determine the underlying influences of RNA/DNA sequences, chromatin environments, three- dimensional proximity and protein interactions on lncRNA nuclear localization. Aim 3: What are the dynamics of FIRRE and CISTR-ACT mediated nuclear organization. We now have adapted CRISPR-Display to Live Cell Imaging (CLING). With this approach we can monitor the 3D interactions of up to 5 chromosomes through time in a living cell. We will investigate the WT and LOF and GOF states of FIRRE and CISTR-ACT chromosomal dynamics in pluripotency and during reprograming. Collectively, these studies will identify the molecular interactions of lncRNAs with DNA and Protein and how they facilitate three and four dimensional organization of the pluripotent genome.