Examining Irf4 genomic programming of lineage development using limited populations of purified immune cells via an optimized protocol for ChIP‐seq (LB176)

Dendritic cell (DC) lineages coordinate immune system activity through functional specialization. Irf4, a transcription factor, is required for CD11b+ DC lineage development from bone marrow stem cells and has been implicated in multiple inflammatory diseases such as asthma. The epigenetic consequences of immune specialization in CD11b+ DCs and relation to inflammatory diseases remain largely unexplored. One reason for this lack of genomic analysis stems from the difficulty of using highly purified, typically limited populations of cells in ChIP‐seq assays. A robust ChIP protocol was developed using limited amounts of K562 cells as a benchmark study. ChIP‐Seq analysis of the transcription factor CTCF or histone modifications strongly correlated with ENCODE data sets. This methodology was used to compare ChIP‐seq analysis of Irf4 genomic binding sites using flow sorted populations of 1, 3, 5, or 25 million CD11b+ lineage DCs. Very similar quality scores and quantitative results comparing Irf4 ChIP‐seq from 5 vs. 25 million sorted CD11b+ DCs were observed. Moreover, results indicate that fewer than 5 million flow sorted cells can be used to acquire high quality ChIP‐seq data. We identified genomic Irf4 binding sites proximal to genes, whose activity is consistent with CD11b+ DC lineage activity and/or known to contribute to inflammatory disease. To examine Irf4 functional regulation of these identified gene targets, RNA‐seq analysis with CD11b+ DCs and a related lineage, CD103+ DCs were performed. We integrated the expression analysis with the ChIP‐seq data and observed a unique CD11b+ DC gene expression program concordant with Irf4 loci association in comparison to CD103+ DC.