The C/EBPα-(PU.1-LOUP) regulatory circuit regulates monocyte/macrophage development and immune functions

The myeloid transcription factors PU.1 and CCAAT/enhancer-binding protein α (C/EBPα) are essential for monocyte/macrophage (Mo/MΦ) development, and their dysregulation has been linked to myeloid malignancies and immune disorders. Although their binding to enhancers for myeloid coding genes is established, their control of noncoding regulatory RNAs remains poorly understood. Using a comprehensive collection of putative and verified enhancers, we profiled the PU.1 cistrome and transcriptome, identifying a subset of noncoding genes that are both associated with PU.1-bound enhancers and regulated by PU.1. Notably, PU.1 induces expression of LOUP, an enhancer RNA transcribed from a locus containing a conserved PU.1 cis-regulatory element cluster, which is characterized by features of myeloid-specific enhancers. Disruption of a PU.1-binding motif in the LOUP promoter and the enhancer reduced LOUP promoter activity, whereas mutation of another PU.1-binding site within the LOUP gene body and the enhancer, which modulates enhancer-promoter interaction, diminished both Pu.1 and Loup levels in mice. The myeloid transcription factor C/EBPα, which binds to the enhancer, is necessary for PU.1 and LOUP expression as inducible deletion of Cebpa in mice led to their downregulation. LOUP depletion impaired Mo/MΦ marker and inflammatory cytokine expression as well as phagocytic function. Collectively, our findings reveal that PU.1 and the enhancer RNA LOUP form a previously unrecognized feed-forward loop, induced by C/EBPα, that drives their mutual expression and establishes a regulatory circuit. This circuit programs monocyte to macrophage differentiation and innate immune function, providing important implications for inflammatory diseases and myeloid malignancies.