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. 2021 Jan 8;10:e60191. doi: 10.7554/eLife.60191

Figure 6. MYC and host cell factor (HCF)–1 bind chromatin independent of their ability to interact.

(A) Scatterplots of normalized average read counts for HCF-1N ChIP-seq peaks in wild-type (WT), 4A, or VP16 HCF-1-binding motif (HBM) switched cells. (B) As in (A) but showing normalized average read counts for MYC–HA ChIP-seq peaks. (C) Heatmap of the combined average normalized peak intensity in 100 bp bins for MYC-HA peaks that were significantly changed (false discovery rate [FDR] < 0.05 and |log2FC| > 0.7) for both the 4A and VP16 HBM mutants, and were within ±2 kb of a TSS. (D) Example IGV screenshots of regions that had significant (top) or non-significant (bottom) changes for MYC-HA or HCF-1N by ChIP-seq. Asterisks mark the peaks that were significantly changed compared to WT. (E) ChIP, using anti-HA antibody, was performed on parental or switchable Ramos cells treated for 24 hr with 20 nM 4-OHT. Enrichment of genomic DNA was monitored by qPCR using primers that amplify across peaks. HBB is a negative locus for HA-MYC. ChIP efficiency was measured based on the percent recovery from input DNA. Shown are the mean and standard error for three biological replicates.

Figure 6—source data 1. MYC-HA ChIP-seq peaks significantly (false discovery rate [FDR] < 0.05) affected by 4A and VP16 host cell factor (HCF)–1-binding motif mutants.

Figure 6.

Figure 6—figure supplement 1. Impact of host cell factor (HCF)–1-binding motif (HBM) mutations on MYC and HCF-1 binding to chromatin.

Figure 6—figure supplement 1.

(A) Recombinant MYC (wild-type [WT], 4A, or VP16 HBM):MAX dimers used in these assays. Dimers were separated by SDS-PAGE alongside a BSA standard and stained using Coomassie Brilliant Blue. (B) Electrophoretic mobility shift assay (EMSA) using recombinant MYC:MAX and MAX:MAX dimers incubated with a biotinylated E-box probe (5′-GCTCAGGGACCACGTGGTCGGGGATC-3′). Binding reactions were conducted with or without 100-fold excess of unlabeled specific (as above) or nonspecific (5′-GCTCAGGGACCAGCTGGTCGGGGATC-3′) competitors. (C and D) Heatmap of the combined average normalized peak intensity in 100 bp bins for all HCF-1N (C) or MYC-HA (D) ChIP-Seq peaks that were within ±2 kb of a TSS, and scaled across each of the cell lines. For clarity in (D), some low-signal data points are excluded. (E) Example IGV screenshots of regions that had significant changes for MYC-HA by ChIP-seq. Asterisks mark the peaks that were significantly changed compared to WT. (F) ChIP, using anti-HCF-1N antibody, was performed on parental or switchable Ramos cells treated for 24 hr with 20 nM 4-OHT. Enrichment of genomic DNA was monitored by qPCR using primers that amplify across peaks. EIF4G3 and HBB are negative loci for HCF-1N. ChIP efficiency was measured based on the percent recovery from input DNA. Shown are the mean and standard error for three biological replicates.