1 Interferon creates transcriptional memory We report that interferon (IFN) stimulation leaves a lasting mark on chromatin over many ISG. This chromatin mark correlated with a memory phonotype: cells that were previously exposed to IFN led to faster and higher ISG induction when restimulated by IFN as compared to nave cells that were not exposed to IFN before. This memory, which we call IFN memory closely resembled a memory response reported in Yeast (Saccharomyces cerevisiae) for genes that respond to nutritional signaling. Type I IFN (IFN alpha and IFN beta)and IFN-gamma both generated memory in fibroblasts and bone marrow derived macrophages, respectively. IFN memory was not attributed to changes in IFN signaling pathways, since levels of IFNAR and phosphorylated STAT1 were similar between nave and memory cells. IFN memory was inherited through several cycles of fibroblast cell divisions. Moreover, this memory gave a functional advantage, in that cells previously exposed to IFN were more effective in the protection against encephalo-myocarditis virus (EMCV) infection. RNA-seq analysis found that about 2,000 ISG induced after IFN stimulation. These ISG were divided into two groups, those with positive memory and those with negative memory. ISG with positive memory showed an elevated response after the 2nd stimulation, while ISG with negative memory were unresponsive or showed reduced response to the 2nd IFN. Positive memory was more prevalent in fibroblasts than negative memory, but in macrophages negative memory was more predominant. Lastly, positive IFN memory correlated with specific chromatin marks, i.e., the acquisition of H3K36me3 mark and H3.3 deposition. Our results illustrate that IFN stimulation creates epigenetic memory by which cells acquire a new, adaptive faculty to respond to changing environments. 2 BRD4 is required for immune responses in a context dependent manner. To study the role of BRD4 in the immune responses we studied ted Brd4 conditional knockout (KO) mice where the Brd4 gene was deleted from the cells of hematopoietic lineage. The initial work found that fetal liver in Brd4 KO mice was grossly abnormal. It had very few HSCs (lineage negative, Sca1+ cKit+ cells) Consequently, Brd4 KO embryos failed to generate erythrocytes, lymphocytes and myeloid cells, and died at around embryonic day 16 to day 17. We concluded that BRD4 is indispensable for embryonic hematopoiesis. However, Brd4 deletion in developing macrophages caused relatively modest defects. Brd4 KO mice generated macrophages with normal surface phenotypes. In vitro deletion of Brd4 from bone marrow had similar results. Furthermore, LPS induction of ISG and other inflammatory factors was only partially reduced in Brd4 KO macrophages, illustrating that BRD4 is largely dispensable for macrophage differentiation and LPS induced inflammatory responses. Nevertheless, BRD4 occupied many genes, over the promoter to the gene body, even on the genes that did not require BRD4 for expression. In addition, BRD4 was broadly present in numerous enhancers both in resting and inflammatory macrophages. The results indicate that there are multiple compensatory mechanisms that cover the lack of BRD4.
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