Control of myeloid lineage fidelity and response to stimuli by ISWI-enforced nucleosome phasing

The interplay between chromatin remodelers and pioneer transcription factors (TFs) regulates cis-regulatory element accessibility to maintain cell identity and transcriptional fidelity. We investigated the impact of imitation of switch (ISWI) chromatin remodelers, key regulators of nucleosome spacing, on macrophage differentiation and activation, focusing on SMARCA5, the sole ISWI ATPase in myeloid cells. Conditional Smarca5 deletion in bone marrow-derived macrophages disrupted nucleosome phasing near sites bound by PU.1, a pioneer TF essential for myeloid identity, without altering PU.1 occupancy. However, SMARCA5 loss increased accessibility at motifs bound by C/EBPβ, a weak pioneer TF, enabling binding to regulatory regions active in non-hematopoietic lineages and causing lineage-inappropriate transcription. These changes also increased accessibility at sites bound by stimulus-induced TFs, leading to macrophage hyperactivation and mis-expression of stimulus-inappropriate genes. Thus, SMARCA5-dependent nucleosome phasing restrains C/EBPβ and stimulus-induced TF binding, ensuring transcriptional fidelity during macrophage lineage specification and activation, with likely similar roles in other immune cell types.