Figure 6.
Figure 6. Regions of differential H3K18Ac are primarily intragenic and are bound by Myc. (A) Ratio heat maps of the level of H3K18Ac from CbpΔ/ΔxEuBcl2 B cells compared with CbpF/FxEuBcl2 B cells from 2 unique biological and technical replicates. These heat maps show the intersection of significant differences identified in each replicate, which includes a small number of regions with significantly reduced peaks of H3K18Ac and a large number of regions with significantly increased peaks of H3K18Ac. (B) Regions of reduced and increased acetylation primarily affect intragenic regions that are distant from the nearest TSS. The distance from the center of the peak of significantly reduced (above) or increased (below) regions of acetylation to the TSS of the nearest gene is shown using a heat plot. It can be seen that the majority of the peaks of differential H3K18Ac lie very far from the nearest TSS, suggesting that they may be distant regulatory elements. (C) An example of motifs that were most significantly enriched in regions of differential H3K18Ac and showed homology to MYC binding sites is displayed. This provided evidence that regions of altered H3K18Ac may be bound by MYC. (D) Public ChIP-seq data for Myc from the Ch12 murine B-cell lymphoma and Mel murine erythroleukemia cell line show a peak of strong Myc binding at the same location as the peak of increased H3K18Ac observed in CbpΔ/ΔxEuBcl2 B-cells. Regions are aligned and show the same physical location as panel A. The average signal (line) ± 1 or 2 standard deviations (shaded region) is summarized at the top of the heat map. The strong peak at the top and Myc binding signal that aligns with the center of the peaks of increased acetylation in our transgenic mice support the binding of Myc to these regions and implicate Myc in the observed epigenetic alterations. (E) Due to the potential importance of Myc in the disease etiology, we confirmed that Myc was also expressed in tumor samples from our transgenic mice. All tumors showed Myc staining, but this was absent from age-matched control spleens. Illustrative examples are shown, with only rare positive cells visible in the control spleen (CbpWT/FxEµBcl2), but broad positive staining in the tumor-involved spleens from mice with 1 or both alleles of Crebbp deleted. Cent., center; cMyc, Myc protooncogene.

Regions of differential H3K18Ac are primarily intragenic and are bound by Myc. (A) Ratio heat maps of the level of H3K18Ac from CbpΔ/ΔxEuBcl2 B cells compared with CbpF/FxEuBcl2 B cells from 2 unique biological and technical replicates. These heat maps show the intersection of significant differences identified in each replicate, which includes a small number of regions with significantly reduced peaks of H3K18Ac and a large number of regions with significantly increased peaks of H3K18Ac. (B) Regions of reduced and increased acetylation primarily affect intragenic regions that are distant from the nearest TSS. The distance from the center of the peak of significantly reduced (above) or increased (below) regions of acetylation to the TSS of the nearest gene is shown using a heat plot. It can be seen that the majority of the peaks of differential H3K18Ac lie very far from the nearest TSS, suggesting that they may be distant regulatory elements. (C) An example of motifs that were most significantly enriched in regions of differential H3K18Ac and showed homology to MYC binding sites is displayed. This provided evidence that regions of altered H3K18Ac may be bound by MYC. (D) Public ChIP-seq data for Myc from the Ch12 murine B-cell lymphoma and Mel murine erythroleukemia cell line show a peak of strong Myc binding at the same location as the peak of increased H3K18Ac observed in CbpΔ/ΔxEuBcl2 B-cells. Regions are aligned and show the same physical location as panel A. The average signal (line) ± 1 or 2 standard deviations (shaded region) is summarized at the top of the heat map. The strong peak at the top and Myc binding signal that aligns with the center of the peaks of increased acetylation in our transgenic mice support the binding of Myc to these regions and implicate Myc in the observed epigenetic alterations. (E) Due to the potential importance of Myc in the disease etiology, we confirmed that Myc was also expressed in tumor samples from our transgenic mice. All tumors showed Myc staining, but this was absent from age-matched control spleens. Illustrative examples are shown, with only rare positive cells visible in the control spleen (CbpWT/FxEµBcl2), but broad positive staining in the tumor-involved spleens from mice with 1 or both alleles of Crebbp deleted. Cent., center; cMyc, Myc protooncogene.

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