Project Summary Chromatin architecture plays important regulatory roles in gene expression and is critical to maintain cellular identity. However, there are many unanswered questions regarding the interplay between chromatin architecture and transcriptional gene regulation, specifically regarding how one influences the other and what factors are involved in orchestrating long-range regulatory chromatin contacts. My proposal attempts to address these questions by focusing on an understudied architectural factor called Ldb1. Ldb1 has been studied at select loci and can facilitate long-range chromatin interactions between regulatory elements and target genes. The direct role of Ldb1 in facilitating chromatin architecture genome- wide to drive lineage-specific gene expression profiles during hematopoiesis has not been clearly defined. We will characterize Ldb1’s role in these processes using a well-studied cellular model system for erythroid differentiation. We will measure genome-wide features of chromatin architecture and Ldb1 genomic occupancy during the dynamic process of erythroid maturation. Additionally, we will acutely deplete Ldb1 to test how and to what extent it is required to maintain chromatin architectural features and gene expression profiles before and after terminal erythroid differentiation. Additionally, we will exploit the natural and highly dynamic processes that occur during cell cycle progression to test Ldb1’s role in establishing chromatin architectural features genome-wide. Mitosis is marked by the eviction of transcription factors, dissolution of most chromatin structure and a global cessation of transcription. During exit from mitosis, daughter cells must re-establish 3D chromatin architecture and transcriptomes that reflect the cell identity of the mother cell. Many features of chromatin architecture arise during mitotic exit through unknown mechanisms and are independent of well-studied architectural factors such as CTCF and Cohesin. We will profile Ldb1 genomic occupancy at closely spaced timepoints during the mitosis-G1 phase transition to determine if its binding dynamics are correlated with the formation of chromatin structures. Furthermore, we will test the requirement of Ldb1 to establish features of chromatin architecture by selectively depleting it in mitosis and measuring cell cycle progression and chromatin architecture reformation genome-wide. By using two natural cell state transitions (erythroid differentiation and cell cycle progression) in addition to acute degradation, we will glean new insights into the underlying mechanisms of chromatin architecture and how they drive lineage-specific gene expression.