The CREBBP-modulated network is enriched in signaling pathways upregulated in the light zone (LZ). The CREBBP-modulated network is enriched in signaling.

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The CREBBP-modulated network is enriched in signaling pathways upregulated in the light zone (LZ). The CREBBP-modulated network is enriched in signaling pathways upregulated in the light zone (LZ). A, Immunohistochemical analysis of Crebbp (top) and Bcl6 (bottom) in representative serial sections from Crebbpfl/flCγ1-Cre (KO) and Crebbp+/+ Cγ1-Cre (WT) mouse spleens, analyzed 10 days after SRBC immunization. B, Western blot analysis of CREBBP and EP300 in splenic GC B cells (B220+CD95+PNAhi) sorted from SRBC-immunized mice (n = 2 mice/genotype, pooled together). BCL6 and actin control for the identity of the fractions and for loading, respectively. C, Heat map of differentially expressed genes in sorted Crebbpfl/fl vs. Crebbp+/+ GC B cells from the Cγ1-Cre cohort (Crebbpfl/+ samples shown by default; Student t test, corrected P < 0.05, FC ≥1.2). Colors visualize differences in the Z score (see scale bar). D, Overlap between CREBBP target genes displaying epigenetic marks of activation and genes downregulated in Crebbp-deficient mouse GC B cells. The complete list of these genes is provided in Supplementary Table S3. The significance of the overlap was calculated by the hypergeometric distribution. E and F, Pathway enrichment analysis of CREBBP core targets (i.e., genes bound by CREBBP in human GC B cells, enriched in active histone marks, and downregulated in Crebbpfl/flCγ1-Cre GC B cells) using the Lymphochip database and MSigDB. Only pathways relevant to B-cell biology are shown in the figure, and the complete lists are available in Supplementary Table S4. Statistical significance was calculated by the hypergeometric distribution and is shown in the color key. G, Gene Set Enrichment Analysis (GSEA) of CREBBP core targets along the T-score rank of transcripts expressed in human dark zone (DZ) vs. LZ GC B cells (top). The reverse analysis (LZ-upregulated genes in Crebbp WT vs. KO GC B cells) is shown at the bottom. H, Leading edge associated with the GSEA shown in G. Selected CREBBP-bound genes with relevant roles in the LZ physiology are indicated. I, ChIP-seq plots of CREBBP and H3K27 binding at PRDM1 and IRF4. Read density tracks of H3K4me1, H3K4me3, and H3K27me3 ChIP-seq enrichment are also illustrated. Boxed areas highlight regions showing significant CREBBP binding. J, Quantification of DZ (Cxcr4hiCd86lo) vs. LZ (Cxcr4loCd86hi) cells ratio in the GC B-cell (B220+CD95+PNAhi) population of Cγ1-Cre and CD19-Cre mice (see Supplementary Fig. S4D and S4H for the gating strategy). In the graph, each dot corresponds to one mouse, and the horizontal bar indicates the mean. WT, Crebbp+/+; HET, Crebbpfl/+; KO, Crebbpfl/fl. Only statistically significant P values are indicated. *, P < 0.05, one-way ANOVA. Jiyuan Zhang et al. Cancer Discov 2017;7:322-337 ©2017 by American Association for Cancer Research