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. Author manuscript; available in PMC: 2014 Feb 15.
Published in final edited form as: J Immunol. 2013 Jan 16;190(4):1827–1836. doi: 10.4049/jimmunol.1201678

FIGURE 5. CD40L/CD40 binding cooperates with IL-21 to load STAT3 onto the shared BCL6/STAT3 site within intron 3 of the PRDM1 gene.

FIGURE 5

A. qRT-PCR analysis of Blimp-1 mRNA in Ly7 cells treated as indicated during a period of 1–4 days. All values were normalized to the pre-treatment level defined as 1.0. B. Schematic representation of the 5′ regulatory region of the human PRDM1 gene and its third intron showing a shared BCL6/STAT3 site. Transcriptional start site +1 is defined as in GenBank accession number AY198414 (http://www.ncbi.nlm.nih.gov/genbank/). Graph is not drawn to scale. C. Ly7 cells pretreated with either control or 3T3-CD40L feeder overnight were left either untreated (ctrl and CD40L) or treated with 100 ng/ml IL-21 for 1 h (IL-21 and IL-21+CD40L). Chromatin was cross-linked and purified from cells subjected to different treatment conditions. Binding of endogenous STAT3 and BCL6 to the indicated regions of the PRDM1 gene were analyzed by qChIP. Signals enrichment by normal rabbit IgG was also measured and used as background control (set as 1.0 in the graphs). D. IL-21/CD40L stimulated transcriptional response from the 3 Blimp-1 Luciferase reporters illustrated at the top. Ly7 cells transiently transfected with the reporters were either left untreated or subject to IL-21/CD40L costimulation as in Figure 3. Cells were harvested for reporter assays 44 h later. Plotted in the graph are the means ± SD of duplicate tests with the mean from the untreated promoter only construct set as 100. E. A working model depicting regulatory relationships between the signaling molecules and transcriptional factors that we have analyzed in this study. Plain lines with arrow heads represent sequential events or positive regulation; blunt bars represent negative regulation. The dashed arrow with a circled plus sign depicts a potentiation effect of activated CD40 on IL-21R/Jak/STAT3 signaling. Roles of the molecules in grey, CD40 and NF-κB, are based on published literature. For simplicity, the direct influence of IRF4 on Blimp-1 as well as inhibitory effects of BCL6 on IRF4 and STAT3 are not shown. PC diff., PC differentiation.