PROJECT SUMMARY/ABSTRACT Dendritic cells are immune sentinels capable of sensing pathogen-derived products and initiating an adaptive immune response. Type 1 conventional DCs (cDC1s) are a specialized subset of DCs that excel in cross- presenting exogenous antigens to CD8+ T cells and secreting interleukin-12, making them an attractive target in evolving therapies for many diseases. Despite the identification of several transcription factors that govern cDC1 biology, little is known about how these transcriptional events are controlled epigenetically to facilitate cDC1 development. In my current research, which serves as preliminary data for this proposal, we determined that optimal cDC1 differentiation, in particular cDC1 terminal maturation and lineage identity, requires cohesin, a complex responsible for establishing the 3D organization of the genome. Thus, the proposed studies to be continued in the independent phase aim to elucidate the chromatin-level control of cDC1 differentiation by cohesin, both globally and at the locus encoding Id2. We will first use scRNA-seq and scATAC-seq in our developed system of cohesin-deficient DC differentiation to precisely define the aberrant differentiation trajectory of cohesin-deficient cDC1s at single-cell resolution and understand the cohesin-dependent transcriptional and chromatin accessibility changes associated with this incomplete and/or divergent trajectory. By integrating these with complementary chromatin conformation capture by Hi-C and CUT&RUN of histone post-translational modifications in cohesin-deficient cDC1s, we will directly test the hypothesis that cohesin- mediated chromatin remodeling is required for the appropriate gene expression program of differentiating cDC1s (Aim 1). We will in parallel investigate the chromatin-level control of the locus encoding Id2, a transcription factor required for cDC1 development (Aim 2). Using CRISPR/Cas9 to engineer mice lacking an architectural element near the Id2 locus