Deciphering the role of IRF4 and its interacting proteins in differentiated regulatory and T helper 17 cells

The transcription factor interferon regulatory factor 4 (IRF4) is crucial for the differentiation and fate determination of pro-inflammatory Th17 and the functionally opposing group of immunomodulatory regulatory T cells.Despite its central role in Th lineage determination, molecular mechanisms of IRF4-mediated gene expression in fully differentiated Th17/Treg cells are still not fully understood. In the present study, we integrated data derived from affinity-purification and full mass spectrometry-based proteome analysis with chromatin immune precipitation sequencing (ChIP-Seq)to unveil IRF4-driven lineage determination in Treg and Th17 cells.This allowed the characterization of IRF4-steered expression of proteins generally involved in the T cell development as well as subtype-specific differentiation and identification of novel, yet uncharted IRF4 interactors. To out knowlede no other study investigated the molecular mechanisms of IRF4-steered gene expression and IRF4 interactions in an unbiased approach, directly comparing fully differentiated T helper 17 and regulatory T cells. Naive CD4+ T cells from splenocytes of IRF4bio (IRF4-Bio.Rosa26BirA) and control (Rosa26BirA) animals were in vitro differentiated into Th17 and Treg cells. IRF4-Bio.Rosa26BirA mice express a fusion protein of IRF4 with a short BirA-recognition site (Avi-Tag, Driegen et al, 2015, PMID: 16201414) and co-express the enzyme BirA, thereby IRF4 gets in vivo biotinylated. Control animals express only the enzyme BirA and have no biotinylated IRF4. After 3 days of cell differentiation with respective cytokines, the cells were harvested and crosslinked with formaldehyde. After cell lysis and sheering of the chromatin, magnetic streptavidin-coated beads (M280 Dynabeads) were added to the lysate and all IRF4-unbound chromatin fragments were washed away. After these extensive washing steps, only sequence-specific IRF4-bound chromatin fragments are eluted and sent for sequencing. As the target protein IRF4 is in vivo biotinylated, the IRF4bio-ChIP approach makes advantage of the strong non-covalent biotin-streptavidin interaction and does not require any IRF4-specific antibodies.