Fig 2: Transcriptional dysregulation in response to Myc and/or Mlx Network inactivation. (A) Gene set enrichment analysis (GSEA) performed on RNA sequencing data sets obtained from MlxKO and DKO livers. Three sets of direct Myc target genes from the MSigDB collection containing 69, 23, and 108 members, respectively, were used in the analysis, with expression levels compared with those of WT livers (N = 5 samples per group). (B) A 154-member collection of direct MondoA, ChREBP, and Mlx target genes from the Qiagen IPA data set was used in GSEA on the samples shown in panel A (Table 2). (C) Volcano plots of differentially expressed genes in the indicated livers expressed relative to WT livers. Reddots, up-regulated; bluedots, down-regulated relative to WT livers. ChrebpKO and MycKO × ChrebKO liver RNA sequencing results were obtained from Wang et al.25 (D) Comparison of MlxKO and DKO livers showing the differential expression of only 14 transcripts. Inset: Heat map of differentially expressed transcripts among WT, MlxKO, and DKO livers. (E) IPA analysis showing the top 7 dysregulated pathways in DKO livers. (F) Heat maps of expression differences for 260 transcripts from the MSigDB C2 database encoding ribosomal subunits and proteins involved in translation (https://www.gsea-msigdb.org/gsea/msigdb/cards/REACTOME_TRANSLATION). (G) Expression differences for 605 transcripts encoding proteins comprising the mitochondrial proteome were compiled from the MitiProteome database (http://www.mitoproteome.org). (H) Myc and Mlx binding sites in HepG2 cells. ChIP sequencing results were downloaded from the Encyclopedia of DNA Elements database (https://www.encodeproject.org) and analyzed for consensus Myc and Mlx binding sites residing within ±2.5 kb of transcriptional start sites. The Venn diagram shows genes that bound only Myc, only Mlx, or both factors. (I) Proximity of Myc and Mlx binding sites within the common target genes shown in panel H. Of the 2433 genes shown, a total of 5267 Myc and Mlx binding sites with overlapping footprints were identified. The positions corresponding to the peak center for each factor and the distances between them were determined. (J) The location and identities of E-boxes and ChoREs in relation to each factor’s binding site peaks. Top: Number of E-boxes and/or ChoREs associated with each footprint. Middle: Proximity of all motifs in relation to their site peak centers (designated as 0). Bottom: Actual location of E-box and ChoRE motifs in each fragment and their position relative to the peak centers. Gray bars correspond to the length (in base pairs) of sequences that were determined. Some genes are depicted more than once because they contained more than a single nonoverlapping binding site. (K) Select functional categories of genes represented by each of the 3 subsets of genes depicted in panel H. Green dotted line: P < .05. (L) The transcriptomes (HTSeq-FPKM-UQ files) of 371 primary HCCs from The Cancer Genome Atlas (TCGA) were downloaded using the TCGAbiolinks R package49 and then assigned to 1 of 4 groups (1–4) based on the expression of the indicated members of the extended Myc Network determined using the ComplexHeatmap R package.50 Tumors within these groups could be assigned to 4 additional categories (A–D) based on the expression patterns of the common 2433 transcripts shown in panel H. A total of 116 of the genes are not shown because their expression was not reported in the TCGA database. (M) Comparative survival of individuals from groups 2 and 4 in panel L.

Fig 3: Characterization of hepatic adenomas originating in MlxKO and DKO livers. (A) Number of mice ages 14–16 months of the indicated genotypes with visible liver tumors at the time of death. (B) Gross appearance of tumor-containing livers from panel A. Tumors were generally small but multifocal (arrows). (C) H&E-stained sections showing the typical appearance of MlxKO and DKO livers. The first 2 MlxKO images show well-differentiated and myxoid-type adenomas, respectively, whereas the third panel shows a focus of well-differentiated HCC embedded within an adenoma. The first 2 DKO images show regions of microvesicular steatosis with balloon cells and nuclear enlargement, respectively, whereas the third image shows another adenoma that is quite similar in appearance to those arising in MlxKO livers. (D) H&E and Ki-67 immunostained sections of the Myc-expressing adenoma from panel D showing regions of inflammation (arrowhead), adjacent to those resembling well-differentiated HCC (arrow). Ki-67 staining is more intense within the nodular adenoma (lower right) compared with adjacent normal liver (upper left). Quantification of Ki-67 staining from 4 different adenomas and adjacent normal tissue was performed on 300–2000 cells from each region. The mean Ki-67 index in adenomas was 30.08% ± 6.5% vs 3.2% ± 3.1% in adjacent nonadenomatous tissues (P = 3.4 × 10-5). (E) Immunoblots showing Mlx expression in WT livers and its absence in adenomas arising from MlxKO and DKO livers. Only 1 large adenoma from an MlxKO mouse with hepatomegaly expressed detectable levels of Myc (lane 2). (F) Principal components analysis (PCA) of whole transcriptomes from WT livers, HBs generated by the overexpression of mutant forms of β-catenin+YAPS127A, and the earlier-described adenomas. (G) Whole-transcriptome profiles of the tissues from panel F. Note that control liver samples were derived from 2 different strains: C57B6 and FVB. (H) Heat maps of 15 of the 22 transcripts that are dysregulated in murine HBs and correlate with poor outcomes in human HBs and other human cancers.47 GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PC, Principal Component.

Fig 4: Targeting strategy and initial reproductive characterization of the MLXKO mice.(A) Cloning strategy for deletion of exons 3 to 6, encoding the DNA-binding domain, of murine Mlx-α with PCR products of the 3 potential genotypes WT, HET, and KO shown to the right. (B) Pups/litter from mating tests between MLX HET males and females (N = 48). (C) Mating test results from crossing the indicated sex and MLX genotype mice (N = 12). (D) Testis weight of WT versus MLXKO mice (N = 7). (E) Epididymis weight of WT and MLXKO mice (N = 5). (F) Body weight of WT and MLXKO mice (N = 7). (G) Histological analysis of WT and MLXKO testis and epididymis stained with hematoxylin and eosin; asterisks mark degenerated seminiferous tubules (100×, scale bar = 400 uM). (H) IF analysis of WT and MLXKO testis and epididymis stained for the indicated protein (100×, scale bar = 400 uM). Shown for all is the mean with SEM with p-values shown from paired t test for groups of 2 and ANOVA for groups of 3 or more (* p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001). The underlying data for Fig 1B–1F can be found in S1 Data. HET, heterozygous; IF, immunofluorescence; KO, knockout; MLX, MAX-Like protein X; WT, wild-type.

Fig 5: MLX and MAX/MDX1 are recruited to Tug1 promoter to coordinate Tug1 repression. A, MLX but not its dominant-negative mutant (DN-MLX) can repress the transcription of Tug1. Cultured mouse podocytes were transfected with Tug1-1kb promoter luciferase construct, together with the indicated Mlx overexpression plasmids or empty vector (Vec) under normal glucose conditions (n = 3). Luciferase readings are presented as mean ± S.E.M. **, p < 0.01; ***, p < 0.001 (compared with vector group); n.s., not significant. B, sequence of glucose-responsive ChoRE and its neighboring E-box in the promoter of mouse Tug1 in WT, ChoRE mutant, E-box mutant, and E-box deletion mutant (ΔE-box). C, both ChoRE (green bar) and E-box (orange bar) bring repressive transcription factors to downregulate Tug1 transcription. Cultured mouse podocytes were transfected with the indicated luciferase constructs of Tug1-1kb promoter (WT), ChoRE and E-box mutants, or empty pGL4 vector (Vec) under normal glucose conditions. Luciferase assays were performed as in A (n = 3). D, MAX and MXD1 can repress Tug1 transcription. Cultured mouse podocytes were transfected with the Tug1-1kb promoter luciferase construct (Tug1-1kb), together with plasmids for the overexpression of the indicated transcription factors or empty pRK5 vector (Vector) under normal glucose conditions. Luciferase assays were performed as in A (n = 3). E, ChIP-qPCR analysis of Tug1 promoter region by the indicated antibodies in podocytes cultured under NG or HG conditions (n = 3). Data are presented as mean ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant (compared with NG group). F, E-box is required for HDAC1-mediated transcription repression of Tug1. Cultured mouse podocytes were transfected with the indicated Tug1-1kb promoter luciferase constructs (WT or E-box mutant) and treated with the indicated concentrations of the HDAC inhibitor TSA or vehicle DMSO for 24 h under normal glucose conditions. Luciferase assays were performed as in A (n = 3).