ABSTRACT Growth, differentiation and survival of immune cells are regulated members of MYC gene family. This family is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and contains three members, the prototype c-MYC (encoded by Myc), N-MYC (Mycn) and L-Myc (Mycl, Mycl1). c-MYC is the most widely used among the many types of immune lineages, but N-MYC is expressed in early hematopoietic stem cells (HSCs), while L-MYC, we discovered several years ago, is expressed in the myeloid subsets of dendritic cells (DCs). We reported that the switch to expression of L-MYC occurs at the stage of the common dendritic cell progeni- tor (CDP), when c-MYC is shut off and L-MYC is induced. We also discovered that L-MYC serves a function in DCs of supporting a robust metabolic phenotype and is required for optimal T cell priming by dendritic cells. The regulation of these various MYC family members is under tight control, but the mechanisms underlying the coordination of their expression is not known. In particular, the mechanism by which c-MYC is repressed is unknown but obviously important at a basic level. In our studies, we have uncovered the fact that the repres- sion of c-MYC at the CDP stage is dependent on the transcription factor IRF8 and that IRF8-deficient mice fail to repress c-MYC and continue to express it in myeloid lineages including classical DCs and plasmacytoid DCs (pDCs). This observation is puzzling because the weight of evidence indicates that IRF8 is an activating tran- scription factor, and no example of direct molecular repression of gene expression is known. To understand how IRF8 can repress c-MYC, we examined the Myc gene locus for IRF8 binding sites by chromatin immune precipitation (ChIP) in a set of progenitor stages of myeloid and DC lineages. We identified several specific regions of IRF8 binding that suggest a concrete hypothesis to explain suppression of c-MYC. These binding sites are located between the c-MYC coding locus and the known Myc enhancer, called BENC, that is located nearly 2 megabases downstream of the Myc gene. IRF8-mediated activation transcription of non-coding RNA that are located between the Myc gene and its enhancer BENC have the potential to alter the chromosomal loop structure of the locus and create a functional blockade preventing access of the Myc gene with its en- hancer, thus causing loss of expression. This R21 application will test this hypothesis directly by deleting the specific IRF8 binding sites specific in primary cells and in vivo using CRISPR/Cas9 methods that we have al- ready established and for which we have a number of published results.