An integrated DNA interactome and transcriptome profiling reveals a PU.1/enhancer RNA-mediated Feed-forward Regulatory Loop Regulating monocyte/macrophage development and innate immune functions

High expression of the myeloid master ETS transcription factor PU.1 drives the development of monocyte/macrophage (Mono/MΦ), a crucial cellular component of the innate immune system. Disruptions in normal expression patterns of PU.1 are linked to a variety myeloid malignancy and immune diseases. It is evidenced that PU.1 binds to and modulates enhancers of several myeloid genes. While noncoding RNAs transcribed from noncoding genes at the enhancers are increasingly reported to be involved in enhancer regulation, the crosstalk between PU.1 and noncoding RNAs in enhancer-mediated myeloid gene regulation in Mono/MΦ differentiation and immune response has not been systematically investigated. In this study, we interrogated the PU.1-mediated transcriptome and cistrome with our comprehensive collection of putative and verified enhancers. Among a repertoire of noncoding genes present at PU.1-bound enhancers, we discovered that PU.1 acts as a potent transcription factor inducer of the noncoding RNA LOUP , which we previously identified as an RNA inducer of PU.1. The genomic region within the LOUP locus occupied by PU.1 is characterized by the epigenetic features of a myeloid-specific super-enhancer. Targeted disruption of the PU.1-binding motifs resulted in the downregulation of LOUP promoter activity. Depletion of LOUP reduced the expression of Mono/MΦ cell markers as well as the transcriptional program associated with Mono/MΦ differentiation Mono/MΦ innate defense mechanisms, including phagocytosis, antimicrobial activity, and chemoattractant cytokine production. LOUP induces Mono/MΦ phagocytic activities. Collectively, our findings indicate that PU.1 and enhancer RNA LOUP are biomolecular components of an unidentified feed-forward loop that promotes their mutual expression, contributing to Mono/MΦ differentiation and innate immune functions. The identification of the PU.1/ LOUP regulatory circuit provides valuable insights into the mechanisms underlying cell-type and gene-specific enhancer activity and Mono/MΦ biology, as well as significant implications for advancing our understanding of immune diseases and myeloid malignancies.