Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal and identity

Tissues in multicellular organisms are 'serviced' by resident macrophages, which perform both generic and tissue-specific functions. The latter relies on unique tissue-specific molecular programs induced by signals from the microenvironment and executed through a combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs) – a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41-deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity. Overall design: RNA-seq comparison of Bhlhe40–/–Bhlhe41–/– and wild-type alveolar (steady-state and from mixed bone marrow chimeras) and peritoneal (steady-state) macrophages (2 biological replicates per condition); H3K27ac ChIP-seq comparison of Bhlhe40–/–Bhlhe41–/– and wild-type alveolar macrophages (steady-state; 2 biological replicates per condition); ChIP-seq characterization of Bhlhe40 DNA binding in WT alveolar macrophages (single experiment) with 'mock' precipitation sample without antibody but with streptavidin sepharose magnetic beads used to generate the control library (single experiment).