PU.1 and IRF8 regulate the transcriptional landscapes differently in naïve and activated B cells

Abstract

Abstract The Ets family member PU.1 and the IRF family member IRF8 play crucial roles in regulating hematopoietic cell development including B cells. Both factors are constitutively expressed at high levels in B cells and control plasma cell differentiation. IRF8 and PU.1 function either together as a heterodimer or by pairing with other factors to bind target DNA sequences. Several consensus binding sequences for IRF8 and PU.1 have been identified, including the canonical interferon stimulated response element (ISRE) sequence motif (GAAANNGAAA), the Ets-IRF composite element (EICE) (GGAANNGAAA) and the IRF-Ets composite sequence (IECS) (GAAANN[N]GGAA). Although the target genes of IRF8 and PU.1 have been studied previously by chromatin immunoprecipitation (ChIP), the nature and the importance of the motifs engaging both PU.1 and IRF8 in B cells have not been determined. In this study, we performed ChIP-seq using sort-purified naïve follicular B cells from C57BL/6 mice that were either not stimulated or stimulated with anti-IgM plus anti-CD40, which mimic signals for germinal center B cell differentiation. We also integrated our ChIP-seq data with RNA-seq data from B cells deficient for both IRF8 and PU.1, and found that IRF8 and PU.1 regulate gene expression by preferentially binding to the EICE motifs of target genes known to maintain follicular B cell identity, localization, and survival. In contrast, in activated B cells, the binding landscape of IRF8/PU.1 in target genes shifts to IRF8-dominant ISRE motifs, possibly due to elevated expression of IRF8. These results provide new insights into our understanding of the molecular mechanisms underlying the transcriptional control of late stage B cell development.