Abstract The spatial organization of chromosomes and gene expression are mutually influential processes. Perturbations of either can lead to developmental defects and disease. Studies on chromatin structure have been dominated by those focusing on the architectural transcription factors CTCF, YY1 and the cohesin complex. The central hypothesis of this application, supported by preliminary data, is that the non-DNA binding adapter protein Ldb1 presents a separate, equally important category of nuclear chromatin organizers. In prior work we demonstrated that artificial recruitment of Ldb1 to chromatin can be sufficient to forge long range enhancer- promoter contacts at the b-globin locus. This nominated Ldb1 as a key architectural protein in erythroid cells. Yet a global view of Ldb1 chromatin occupancy patterns during cell differentiation or cell cycle progression is lacking, and genome wide direct Ldb1 dependent chromatin contacts and corresponding transcriptional patterns are largely unknown. More broadly, the cause-effect relationships of architectural features and gene expression are still hotly debated in the field. Here we propose to carry out the following aims in collaboration with the laboratory of Dr. Ross Hardison. In Aim 1 we will assess in erythroblasts undergoing cell maturation the dynamic Ldb1 chromatin landscapes in relation to high resolution Hi-C chromosomal connectivity maps, and nascent transcription measurements (PRO- seq). In Aim 2 we will use an auxin-inducible acute Ldb1 degradation system to interrogate direct architectural and transcriptional Ldb1 dependencies. In Aim 3 we will exploit the massive and swift changes in chromatin architecture during the mitosis to G1-phase progression to assess the chromatin occupancy dynamics of Ldb1 and its role in establishing the multi-layered hierarchy of chromatin organization and gene expression. All aims are accompanied by perturbative and mechanistic experiments. We believe that the power of the proposed studies lies in the complementarity of highly dynamic natural transition states (cell maturation and cell cycle progression) as well as acute experimental perturbation (auxin degron), which to our knowledge has never been done before within the same cell system. Gains in knowledge are further enhanced by parallel studies in the same cellular system on CTCF, YY1 and cohesin with support from different funding sources. The forward looking view is that the end result of our studies is, for the first time, a unified view of the dynamics of major architectural components and gene expression. Therefore, the studies proposed here coupled with parallel studies in the lab generate a cost-effective synergy, such that the outcome will be greater than the sum of its parts.