Fig 2: Differential MAX, MGA, and MNT chromatin binding alterations in MYCi975-treated cells.(A) Peak overlap analysis of MYC and MAX consensus peaks (n = 4). (B) Percentage of differential MAX binding peaks lost in response to MYCi975 compared to MAX consensus peaks (n = 4). (C) Total number of differentially bound MAX peaks following differential binding analysis (n = 4). (D) Peak overlap analysis of differential MYC- and MAX-bound sites (MYCi975 sensitive) in MYCi975-treated 22Rv1 cells. (E) Heatmap representation of MYC, MAX, MGA, MNT, and MXD1 at MYCi975-sensitive sites demonstrating no significant change in MAX occupancy (6812 peaks in total). (F) Log2(normalized tag counts) for MYC, MAX, MGA, MNT, and MXD1 at MAX-retained peaks in 22Rv1 cells. The middle dashed line represents the sample median, and the upper and lower dotted lines represent the upper and lower quartiles, respectively. (G) Differential gene expression analysis of MAX-retained peaks annotated to promoters (±2 kb from the TSS). (H) Gene ontology analysis of promoter-bound MAX-retained peaks. (I) Gene browser tracks of AURKB showing loss of MYC (green), retention of MAX (blue), an increase in MXD1 (red), and loss of AURKB mRNA (black) in 48-hour MYCi975-treated cells. MGA (gray) and MNT (yellow) are also displayed.

Fig 3: MLX shares transcriptional targets with MAX and MNT.(A) Meta-plots of TSS occupancy for the indicated TFs in WT 3T3 cells. (B) E-Box Motif derived from HOMER analysis with the indicated p-value. (C) Venn diagram indicating overlap of genes bound by MLX, MAX, and MNT in 3T3 cells with p-value calculated from a hypergeometric test. (D) ChIP-Seq tracks for MLX, MAX, and MNT on the indicated gene promoters from WT and MLXKO 3T3 cells. (E) WBs from WT and MLXKO and WT and MNTKO littermate 3T3 cell lines probed for the indicated proteins. (F) Diagram summarizing modes of MondoA, MNT, and MLX association and consequent transcriptional responses. The underlying data for Fig 8A–8D can be found in S1 Data. ChIP-Seq, chromatin immunoprecipitation and sequencing; E-box, Enhancer box; KO, knockout; MLX, MAX-Like protein X; TF, transcription factor; TSS, transcription start site; WB, western blot; WT, wild-type.

Fig 4: Alterations in MNT genomic occupancy correlates with gene expression changes in RP and RPMax tumor cells.A) Plots of genomic region over gene bodies (x-axis) vs normalized coverage (y-axis) of MAX/MNT bound cluster 1 (see Figure S3C), which was a cluster of peaks determined to be differentially occupied by MNT. Coverage of MAX for RP (MAX WT, blue) is compared to that from RPMax (MAX KO, red) cells. B) Plots of MNT coverage of MAX/MNT bound cluster 1 comparing RP (blue) and RPMax (red) cells, similar to panel A. C) Genomic tracks showing differential occupation of the Rorc promoter in RP (Max WT) and RPMax (Max KO) cells as determined by ChIP-Seq. The antibodies (against MAX, MNT, MLX) used for ChIP are shown (at left) after the hyphen. D) Volcano plot of RNA-Seq gene expression changes comparing RP vs RPMax thyroid tumors (Log2FoldChange on the x-axis, −Log10 adjusted p value on y-axis). Genes with peaks that map to MAX bound sites (blue) and in MAX/MNT cluster 1 (red) are shown. E) Cumulative distribution plots depicting the ranked-order (y-axis, Percent of genes) vs log2FoldChange (x-axis) of RNA-Seq data (comparing RPMax vs RP thyroid tumors). MNT cluster 1 genes (dark blue) and a similarly sized set of genes determined to not be MNT occupied (light blue) are shown. The difference between the distributions is statistically determined using Kolmogorov-Smirnov (KS) statistics. F) Enrichment analysis of genes found to be differentially occupied by MAX and MNT in Max WT RP tumor-derived thyroid cell lines (Mnt cluster1 from Fig. S5C). Enrichment categories are plotted as the −log10 of the adjusted P value (y-axis, −log10 padj), and Odds.Ratio (x-axis).

Fig 5: Differential sensitivity and changes in gene expression of Max-inactivated thyroid tumor upon inhibition of MNT, MLX and MondoA.A) Thyroid tumor cell lines (3 independent RP and 3 RPMax lines) were transfected with the indicated siRNA and allowed to reconstitute over 4 days. Outgrown spheres were enumerated and normalized to the siCtrl condition. siDeath is included as a control for transfection efficacy. B) Thyroid tumor cell lines (3 independent RP and 3 RPMax lines) were seeded at equal density with either vehicle (DMSO) or SBI-477 (Mondo Inhibitor, 20uM). After 4 days, cells were counted and normalized to DMSO. C) RPMax thyroid tumor cell line H7536, reconstituted with either control vector (pCW Vector) or doxycycline-inducible Max construct (pCW MAX) were cultured in the absence or presence of DOX and/or SBI-477 for 96 hours then cells were counted and normalized to untreated cells for each line. For all data, p-values were calculated using ANOVA with a Tukey’s adjustment. D) Enrichment analysis of genes that were down-regulated in tumor cells expressing wild-type Max, but up-regulated in tumor cells with Max deleted upon MondoA silencing (using a Mondo inhibitor, SBI-477). Enrichment categories are plotted as the −log10 of the adjusted P value (y-axis, −log10 padj), and Odds.Ratio (x-axis). E, F) Volcano plots of RNA-Seq gene expression changes (Log2FoldChange on the x-axis, −Log10 adjusted p value on y-axis) comparing cells treated with vehicle control to cells treated with MondoA inhibitor (SBI-477) for RP cells (D) or RPMax cells (E). Genes that are important in the unfolded protein response are highlighted in blue. G) Plot of expression changes (Log2FoldChange) of RP (y-axis) vs RPMax (x-axis) cells. Genes important for the unfolded protein response are highlighted by yellow-red color. Yellow-red shading depicts the adjusted p value (padj), and higher red intensity indicates greater significance in gene expression change between the two datasets.