Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4

The transcription factor IRF4 regulates immunoglobulin class switch recombination and plasma cell differentiation. Its differing concentrations appear to regulate mutually antagonistic programs of B and plasma cell gene expression. We show IRF4 to be also required for generation of germinal center (GC) B cells. Its transient expression in vivo induced the expression of key GC genes including Bcl6 and Aicda. In contrast, sustained and higher concentrations of IRF4 promoted the generation of plasma cells while antagonizing the GC fate. IRF4 cobound with the transcription factors PU.1 or BATF to Ets or AP-1 composite motifs, associated with genes involved in B cell activation and the GC response. At higher concentrations, IRF4 binding shifted to interferon sequence response motifs; these enriched for genes involved in plasma cell differentiation. Our results support a model of "kinetic control" in which signaling-induced dynamics of IRF4 in activated B cells control their cell-fate outcomes. Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request ([e-mail]) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format. ChIP was performed by using anti-IRF4, -PU.1, and control IgG antibodies (Santa Cruz Biotech. and Abcam) (Sciammas et al., 2006). For massively parallel sequencing, 200 μg of chromatin fragments were immunoprecipitated by using anti-IRF-4 and anti-PU.1 antibodies, and DNA libraries were prepared. DNA was sequenced by using the Illumina GA2, data was processed with the Solexa pipeline package, and sequences were aligned to the mouse genome (mm9) by using ELAND software. For transcriptome analysis, total RNA was prepared from triplicate cell samples by using Trizol and processed for hybridization to Affymetrix mouse 430A chips by using standard procedures. For qRT-PCR analysis of sorted SWHEL B cells, total RNA was prepared by sorting 5,000 cells directly into RLT buffer from the RNeasy Micro Kit (QIAGEN). More details are provided in the supplementary information (web link) and in the manuscript.