Partner-regulated interaction of IFN regulatory factor 8 with chromatin visualized in live macrophages

IFN regulatory factor (IRF) 8 is a transcription factor that directs macrophage differentiation. By fluorescence recovery after photobleaching, we visualized the movement of IRF8-GFP in differentiating macrophages. Recovery data fitted to mathematical models revealed two binding states for IRF8. The majority of IRF8 was highly mobile and transiently interacted with chromatin, whereas a small fraction of IRF8 bound to chromatin more stably. IRF8 mutants that did not stimulate macrophage differentiation showed a faster recovery, revealing little interaction with chromatin. A macrophage activation signal by IFN-gamma/LPS led to a global slowdown of IRF8 movement, leading to increased chromatin binding. In fibroblasts where IRF8 has no known function, WT IRF8 moved as fast as the mutants, indicating that IRF8 does not interact with chromatin in these cells. However, upon introduction of IRF8 binding partners, PU.1 and/or IRF1, the mobility of IRF8 was markedly reduced, producing a more stably bound component. Together, IRF8-chromatin interaction is dynamic in live macrophages and influenced by partner proteins and immunological stimuli.