Achim Leutz

The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of myelopoiesis. CEBPA encodes a long (p42) and a truncated (p30) protein isoform from a single mRNA. Mutations that abnormally enhance expression of p30 are associated with acute myelogenous leukemia (AML). We show by mutational analysis that three highly conserved arginine residues (R140,147,154) located at the p30 C/EBPα N-terminus, previously found to be methylated, are involved in myeloid lineage commitment, progenitor proliferation, and differentiation. Replacement with lysine that retains the amino acid side chain charge enhanced progenitor proliferation, while uncharged side chains (alanine or leucine) impaired proliferation and enhanced granulopoietic differentiation. Analysis of protein-protein interactions (PPI) suggested that arginine methylation of p30 C/EBPα differentially determines its capacity to interact with SWI/SNF and MLL complexes. Pharmacological targeting of p30 C/EBPα arginine methylation may have clinical relevance in myeloproliferative and inflammatory diseases, in neutropenia, and in leukemic stem cells.

Cell fate decisions are driven by lineage-restricted transcription factors but how they are regulated is incompletely understood. The C/EBPα-induced B cell to macrophage transdifferentiation (BMT) is a powerful system to address this question. Here we describe that C/EBPα with a single arginine mutation (C/EBPα ^R35A ) induces a dramatically accelerated BMT in mouse and human cells. Changes in the expression of lineage-restricted genes occur as early as within 1 hour compared to 18 hours with the wild type. Mechanistically C/EBPα ^R35A exhibits an increased affinity for PU.1, a bi-lineage transcription factor required for C/EBPα-induced BMT. The complex induces more rapid chromatin accessibility changes and an enhanced relocation (stealing) of PU.1 from B cell to myeloid gene regulatory elements. Arginine 35 is methylated by Carm1 and inhibition of the enzyme accelerates BMT, similar to the mutant. Our data suggest that the relative proportions of methylated and unmethylated C/EBPα in a bipotent progenitor can determine the velocity of cell fate choice and lineage directionality.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPβ-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPβ protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPβ as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1 ⁺ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2 ⁺ macrophages were detected in deeper muscular tissue. The two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo . Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The pioneering transcription factor C/EBPα coordinates cell fate and cell differentiation. C/EBPα represents an intrinsically disordered protein with multiple short linear motifs and extensive post-translational side chain modifications (PTM), reflecting its modularity and functional plasticity. Here, we combined arrayed peptide matrix screening (PRISMA) with biotin ligase proximity labeling proteomics (BioID) to generate a linear, isoform specific and PTM-dependent protein interaction map of C/EBPα in myeloid cells. The C/EBPα interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport as the basis of functional C/EBPα plasticity. Protein interaction hotspots were identified that coincide with homologous conserved regions of the C/EBP family and revealed interaction motifs that score as molecular recognition features (MoRF). PTMs alter the interaction spectrum of multi-valent C/EBP-motifs to configure a multimodal transcription factor hub that allows interaction with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID acts as a powerful strategy to systematically explore the interactomes of intrinsically disordered proteins and their PTM-regulated, multimodal capacity. Key points Integration of proximity labeling and arrayed peptide screen proteomics refines the interactome of C/EBPα isoforms Hotspots of protein interactions in C/EBPα mostly occur in conserved short linear motifs Interactions of the BAF/SWI-SNF complex with C/EBPα are modulated by arginine methylation and isoform status The integrated experimental strategy suits systematic interactome studies of intrinsically disordered proteins