Figure 1. A gain- and loss-of-function system to study the MYC–host cell factor (HCF)–1 interaction.

(A) Schematic of MYC, depicting the location of the six MYC boxes (Mb0–MbIV). MbIIIb carries a WDR5-binding motif (WBM). MbIV contains an HCF-1-binding motif (HBM). Residues relevant to the WBM or HBM are in bold, and residues mutated in this study are in red. (B) FLAG-tagged full-length MYC proteins carrying the mutations described in (A) were transiently expressed in 293T cells, recovered by anti-FLAG immunoprecipitation (IP), and the input, or IP eluates, probed for the presence of HCF-1_C, WDR5, or FLAG-tagged proteins by western blotting. (C) Western blot of lysates from parental (CRE-ER^T2) or switchable Ramos cells (wild-type [WT], 4A, or VP16 HBM) ±20 nM 4-OHT for 24 hr. Blots were probed with antibodies against the HA tag, MYC, HCF-1_C, and GAPDH. (D and E) Parental or switchable Ramos cells (WT, 4A, or VP16 HBM) were treated with 20 nM 4-OHT for 24 hr, lysates prepared, and IP performed using anti-IgG or anti-MYC antibodies. Input lysates and IP eluates were probed using antibodies against HCF-1_C, WDR5, HA tag, and MYC by western blotting. All lines in these experiments express CRE-ER^T2. (F) Switchable Ramos cell lines were pulsed with 20 nM 4-OHT for 2 hr to switch ~50% of cells, propagated for 3 days, and grown for 16 hr in media with or without glutamine. The impact of glutamine deprivation was measured by flow cytometry to determine the proportion of green fluorescent protein (GFP)-positive (switched) cells. For each of the mutants, the proportion of GFP-positive cells was normalized to that for WT cells. Shown are the mean and standard error for three biological replicates. Student’s t-test between +Gln and −Gln was used to calculate p-values; a = 0.0066, b = 0.0002. (G) Switchable Ramos cells were pulsed with 4-OHT as in (F), grown for 7 days, and cell cycle distribution determined by propidium iodide (PI) staining and flow cytometry, binning cells according to whether they expressed GFP (GFP+, switched) or not (GFP−, unswitched). Shown are the mean and standard error for three biological replicates. Student’s t-test between GFP− and GFP+ cells was used to calculate p-values; a = 0.033, b = 0.0041, c = 0.0006. (H) Switchable Ramos cells were pulsed with 4-OHT as in (F), and the proportion of GFP-positive cells measured by flow cytometry 24 hr after treatment and every 3 days following. For each of the replicates, the proportion of GFP-positive cells is normalized to that on day 1. Shown are the mean and standard error for three biological replicates. Student’s t-test between WT and each of the mutants at day 25 was used to calculate p-values; a = 0.000028, b = 0.00026.

Figure 1—figure supplement 1. Validation of MYC mutants and switchable Ramos cell lines.

(A) Recombinant FLAG-tagged MYC was purified from E. coli (Rosetta) cells by nickel affinity chromatography. Shown are proteins from two sequential elutions with imidazole-containing buffer (E1 and E2), which were resolved by SDS-PAGE alongside a bovine serum albumin (BSA) standard and detected by Coomassie staining. (B) In vitro transcribed/translated T7-tagged host cell factor (HCF)–1_VIC was incubated with recombinant FLAG-tagged MYC, either wild-type (WT) or mutant (4A or VP16 HCF-1-binding motif [HBM]), and IP performed using anti-FLAG M2 agarose. Western blot of the input lysate, and the IP eluate, was probed using antibodies against the T7 and FLAG tags. (C) The translocated MYC locus from Ramos cells is depicted at top, with chromosome 14 (red) and 8 (blue) elements indicated. Beneath is a representation of the locus modification, in either the unswitched (middle) or switched (bottom) states. This switchable allele contains a WT exon 3, a P2A-linked puromycin cassette, and a SV40 polyadenylation (SV40 PA) signal, all of which are flanked by LoxP sites (black triangles). Downstream of the LoxP-flanked region is an HA-tagged mutant exon 3 (mut-Ex3) and a P2A-linked green fluorescent protein (GFP) cassette, with the endogenous 3’ untranslated region (UTR) intact. Activation of CRE-ER^T2 results in excision of WT exon 3 and its replacement with mutant exon 3 which carries sequences encoding either WT or mutant (4A or VP16 HBM) MYC protein. (D) Comparison of the structure of the parental (non-modified) MYC allele (top) compared to the switchable MYC allele (bottom). XbaI sites used for digestion of genomic DNA in Southern blot are highlighted, as are the complementary sites for the MYC and GFP probes. The expected products of XbaI digestion for the parental line are 6784 bp for the MYC probe (which detects both the translocated and non-translocated alleles) and nothing for the GFP probe; for correctly engineered lines the expected sizes are 6784 bp and 2942 bp for the MYC probe, and 6624 bp for the GFP probe. (E) Southern blot using GFP and MYC probes on XbaI-digested gDNA from unswitched parental or switchable cells (WT, 4A, and VP16 HBM), with digested positive and negative control plasmids. (F) Switchable cells were treated with or without 20 nM 4-OHT (24 hr), fixed using 1% formaldehyde, and subject to flow cytometry. The GFP profiles of the −4-OHT and +4-OHT cells are shown overlaid onto the same axes, with the approximate percentage of GFP-positive cells for 24 hr +4-OHT shown.

Figure 1—figure supplement 2. Localization of MYC mutants and their impact on cell doubling time.

(A) Chromatin fractionation of switchable Ramos cells was performed after a 24 hr treatment with 20 nM 4-OHT. S2 reflects the cytosolic, S3 the non-chromatin nuclear, and P3 the chromatin-bound fraction. Each fraction was probed using antibodies against host cell factor (HCF)–1_C, HA, α-Tubulin, and H3. (B) Immunofluorescence of cells that had been switched for 24 hr as in (A), using a DAPI DNA stain and an antibody against the HA tag on switched MYC proteins. (C) Switchable Ramos cell lines were treated with 20 nM 4-OHT for 16 hr, then re-plated 24 hr later at a density of 2 × 10⁴ cells/ml. The number of cells, including both green fluorescent protein (GFP)– and GFP+, was counted at 3 and 6 days following re-plating. Shown are the mean and standard error for three biological replicates. Student’s t-test between wild-type (WT) and mutant cells was used to calculate p-values; **=0.0023, ***=0.00087. (D) Using the difference in cell number between day 1 and day 7 from (C), we calculated growth rate and subsequently the approximate doubling time for each cell line following switching. Bar graph shows the mean and standard error for the three biological replicates. Student’s t-test between WT and mutant cells was used to calculate p-values; a = 0.002, b = 0.0012. Table below shows the mean growth rates and doubling time ± standard deviation.