Fig 2: ZMYND8 Deficiency Causes Defective Germline Transcription of Acceptor S RegionsqPCR analysis for Aicda mRNA, Igµ, and Ig?1 (A), Ig?2b (B), and Ig?3 (C) GLT levels in B cells activated to undergo CSR to the corresponding isotypes. The schematic representations indicate the location of primers employed to analyze pre- and post-spliced germline transcripts. One Cd19Cre/+ mouse within each experiment was assigned an arbitrary value of 1 (error bars represent SD). Significance was calculated with the Mann-Whitney U test. **p = 0.01, ***p = 0.001. See also Figure S5.

Fig 3: Model for ZMYND8 Function in Igh Gene DiversificationZMYND8 controls both CSR and Igh SHM via its ability to regulate the activity of the 3' Igh super-enhancer. GTL for S-C?1 is representative of acceptor S region induction.

Fig 4: ZMYND8 Represses 3'RR Enhancer Transcription(A) (Top) ZMYND8 and Pol II loading at the Igh locus in splenocytes stimulated for 72 hr with LPS and IL-4. (Bottom) Pol II ChIP-seq tracks overlay at regions encompassing Eµ and 3'RR enhancers.(B) Pol II loading quantification at Igh enhancers. Graph summarizes ChIP-seq data from three mice per genotype (error bars represent SD). Significance was calculated with Welch 2 sample unpaired t test.(C) qPCR analysis for hs1,2 and hs3b eRNA levels in B cells 48 hr after stimulation with LPS and IL-4 (left) or LPS only (right). Two sets of primers amplifying the 5' (5' hs3b) and 3' (3' hs3b) regions of hs3b were employed. The data summarize 4–6 mice per genotype (error bars represent SD). One Cd19Cre/+ mouse within each experiment was assigned an arbitrary value of 1.0. Significance was calculated with the Mann-Whitney U test.(D) (Left) Heatmap showing Igh 3'RR enhancers differential transcript expression as determined by RNA-seq in controls (WT bulk and clonal derivative WTc) and two independent Zmynd8-/- CH12 clones (KO1 and KO2). Expression counts are row-normalized by Z score. Three independent RNA-seq replicates per sample are shown. (Right) Bar graphs depicting relative transcript levels at hs4, hs1,2, hs3b, and hs3a are shown. The adjusted p values were calculated with the Wald test and corrected for multiple testing with the Benjamini-Hochberg method.(E) (Left) Graph depicting ZMYND8-bound B cell super-enhancers that are differentially expressed in Zmynd8-/- versus WT CH12 as measured by RNA-seq. DE SE, differentially expressed SE. (Right) Plot of fold change of Pol II loading on the differentially expressed super-enhancers defined in the left panel is shown.**p = 0.01. See also Figure S7 and Table S4.

Fig 5: ZMYND8 Binds Promoters and Super-Enhancers in B Cells(A) Genomic distribution of ZMYND8 ChIP-seq peaks in CH12 cells.(B) Venn diagram of the overlap between ZMYND8 peaks and B cells super-enhancers. SE, super-enhancer.(C) ZMYND8 occupancy at the Igh locus in WT and Zmynd8-/- CH12 cell lines (KO1 and KO2). A schematic representation of the murine Igh locus showing location of enhancers (in gray) and S regions (in black) is represented below.Data in (A)–(C) are representative of two independent experiments.