This laboratory is interested in chromatin regulated gene expression with an emphasis on innate immunity. We currently focus on two nuclear regulatory factors Brd4 and IRF-8. Brd4 is a mammalian bromodomain protein that interacts with acetylated chromatin. IRF-8 is a DNA binding transcription factor of the IRF family important for innate immunity. Our goal is to elucidate the mechanism by which these two factors act on chromatin and regulate transcription in living cells.
To study binding of IRF-8 to chromatin in live macrophages, we have employed the fluorescence recovery after photobleaching method. This method allows us to visualize dynamic movement of transcription factors and their real time interactions with chromatin in living cells. We found that the majority of IRF-8 is moving extremely fast, binding to chromatin for less than 0.1 second and then moving away to bind again elsewhere. Only about 10% of IRF-8 showed more stable interaction. A macrophage activating stimulus (IFN g /LPS) markedly increased the more stably bound fraction, indicating that an immunological stimulus can change a global behavior of IRF-8. We found that interaction of IRF-8 with chromatin was contingent upon its interaction with the partners, PU.1 and IRF-1. Together, these results are consistent with the idea that transcription factors are interact with chromatin only transiently, constantly scanning the entire genome. Live cell technologies such as photobleaching open a new horizon to our understanding of nuclear events and gene expression.
Figure 1. IRF8-GFP induced macrophage differentiation in Tot2 progenitor cells, but the mutant IRF8-GFP did not. FRAP analysis showed that wild type IRF8 binds to chromatin with characteristic fast on-and-off kinetics, but the mutants showed no sign of chromatin binding.
Figure 2. Brd4 and Brd2 belong to the BET family and have two bromodomains through which they interact with acetylated chromatin. They remain associated with chromatin during mitosis. The lower panel shows immunostaining of endogenous Brd4 on spread chromosomes.
Figure 3. An equilibrium model for Brd4-P/TEFb interactions. P-TEFb occurs either complexed with Brd4 or the inhibitory subunit. The Brd4 bound P-TEFb is recruited to a promoter in acetylated chromatin and stimulates RNA polymerase II dependent transcription.
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