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, 293 (38), 14740-14757

Myc and ChREBP Transcription Factors Cooperatively Regulate Normal and Neoplastic Hepatocyte Proliferation in Mice

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Myc and ChREBP Transcription Factors Cooperatively Regulate Normal and Neoplastic Hepatocyte Proliferation in Mice

Huabo Wang et al. J Biol Chem.

Abstract

Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element-binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in myc-/-, chrebp-/-, or myc-/-/chrebp-/- backgrounds grew significantly more slowly. Metabolic studies on livers and HBs in all three genetic backgrounds revealed marked differences in oxidative phosphorylation, fatty acid β-oxidation (FAO), and pyruvate dehydrogenase activity. RNA-Seq of livers and HBs suggested seven distinct mechanisms of Myc-ChREBP target gene regulation. Gene ontology analysis indicated that many transcripts deregulated in the chrebp-/- background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the myc-/- background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the myc-/-/chrebp-/- background, additional deregulated transcripts included those involved in peroxisomal β- and α-FAO. Finally, we observed that Myc and ChREBP cooperatively up-regulated virtually all ribosomal protein genes. Our findings define the individual and cooperative proliferative, metabolic, and transcriptional roles for the "Extended Myc Network" under both normal and neoplastic conditions.

Keywords: MYC proto-oncogene bHLH transcription factor; MondoA; Myc (c-Myc); Oxphos; Warburg effect; fatty acid oxidation; hepatoblastoma; hepatocellular carcinoma; liver cancer; mitochondria.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Competitive disadvantage of ChREBP-KO and Double-KO fah+/+ donor hepatocytes. A, basis of the FAH competitive repopulation assay. GFP+ fah+/+ hepatocytes were used as the control, WT donor population. They were mixed in approximately equal numbers with ChREBP-KO or Double-KO hepatocytes (both GFP− and fah+/+). Following intrasplenic injection of the mixed hepatocyte population into fah−/− mice, animals were maintained on NTBC for 4 days and then periodically cycled off and on the drug until weights stabilized. Hepatocytes were then isolated, and the relative proportions of donor and recipient populations were determined. B, TaqMan primer set used to quantify the proportion of total fah−/− recipient, post-transplant hepatocyte population. Primers were designed to amplify the flanking region of the neo cassette-exon 5 region of the fah−/− recipient population. C, reconstitution of livers following transplantation of a donor population composed of WT + ChREBP-KO hepatocytes (top) or WT + Double-KO hepatocytes (bottom). Total donor and recipient populations from 8–10 transplanted mice were quantified as described in B. Numbers to the right indicate the final mean percentage repopulation by the total donor population. D, TaqMan primers used to distinguish each of the two donor populations. The primer set used to amplify GFP+ WT donor hepatocytes amplified a region of the GFP cassette. The primer set used to amplify the ChREBP-KO donor population amplified the region flanking the neo cassette and exon 14 of the chrebp gene. The primer set used to detect the Double-KO population amplified a region of Cre recombinase, which was present only in the Myc-KO population. E, hepatocyte DNAs from C were used as templates for the TaqMan primers depicted in D to allow a determination of the relative proportion of WT and ChREBP-KO donor hepatocytes (top) or WT + Double-KO donor hepatocytes (bottom). The first bar (input) indicates the percentage composition of the initial input population. Numbers to the left indicate the percentage of WT hepatocytes in input or transplanted hepatocyte populations. The values for C and E were obtained from reconstitution experiments using eight different known ratios of each of the two DNA populations being quantified as described previously (51). Correlation coefficients in each case were >0.99 (not shown).
Figure 2.
Figure 2.
Behaviors and properties of WT and KO tumors. A, Kaplan–Meir survival curves of the indicated groups of mice following hydrodynamic tail vein injection of plasmids encoding mutant forms of β-catenin and YAP (13, 27) (n = 10–15 mice/group). B, tumor weights at the time of sacrifice in each group. Note that tumor weights in all three KO groups (Myc-KO (MKT), ChREBP-KO (CKT), and Double-KO (DKT)) were assessed much later than those of WT tumors. C, expression of proliferating cell nuclear antigen (PCNA) and phosphohistone H3 in livers (L) and tumors (T) from the indicated tissues. D, transcript levels of members of the Extended Myc Network. Also included is the expression of genes (myct1/mt-mc1, hmga1, and shmt) that can rescue the growth defect of myc−/− fibroblasts (57, 59). Relative mRNA expression levels of the indicated genes were determined directly from RNA-Seq results (n = 5 samples/group). The numbers within boxes indicate the relative expression of each transcript. Myc transcript levels in Myc-KO and Double-KO livers and tumors and ChREBP transcripts in ChREBP-KO and Double-KO livers and tumors arise almost exclusively from transcripts upstream of the neo cassettes that disrupted the coding region of the genes (not shown). E, expression of select members of the Extended Myc Network in representative livers and HBs from each of the four cohorts. Two sets of immunoblots (Set 1 and Set 2) are depicted (see Fig. S2 for two additional sets). Error bars, S.D.
Figure 3.
Figure 3.
Metabolic differences distinguish WT, Myc-KO, ChREBP-KO, and Double-KO livers and tumors. A, liver and tumor OCRs. The indicated tissues were dispersed, and their maximal OCRs were determined by respirometry following the addition of the TCA cycle substrates pyruvate, malate, and succinate. Complex I and Complex II responses were distinguished by measuring OCR before and after the addition of the Complex I inhibitor rotenone. B, reduction of mtDNA content in HBs is independent of tumor growth rates and the presence of Myc and/or ChREBP. Two different sets of TaqMan probes (Set 1 and Set 2) were used to independently amplify different regions of the mitochondrial genome. C, ATP levels in livers and tumors. D, AcCoA levels in livers and tumors. E, PDH activity in fresh livers and tumors. PDH was assessed by measuring the release of 14CO2 from 14C-labeled pyruvate (13, 51). F, FAO rates in fresh livers and tumors. FAO was assessed by measuring the release of water-soluble products from 3H-labeled palmitate-BSA (13, 51). G, representative Coomassie Blue–stained nondenaturing gels of ETC complexes from livers (L) and tumors (T) isolated from the indicated groups. The panel shows two sets of results. H, typical appearances of developed nondenaturing gels following in situ enzymatic assays for the ETC complexes I, III, IV, and V. I, quantification of ETC complex activities. Four sets of gels similar to those shown in H were scanned and normalized to the activity of Complex IV, which showed little variation among the different tissues (13, 25, 105). J, complex II activities were assayed in vitro on tissue lysates because the in situ assay is unreliable. All results were normalized to the mean activity of each complex measured in WT livers. Error bars, S.D.
Figure 4.
Figure 4.
Summary of observed gene expression changes in WT, Myc-KO, ChREBP-KO, and Double-KO livers and tumors. A, Venn diagram summarizing the relationships of transcriptional profile differences among livers and HBs. All transcripts could be grouped into one of seven categories (1–7) based on which gene expression differences were shared between or among different groups. B, potential mechanistic explanations for the types of gene expression changes in the groups described in A, assuming Myc and/or ChREBP act as direct positive or negative regulators. The models outlined here are meant to be heuristic in nature and not necessarily inclusive of all members of the indicated group. Group 1 (Dual-dependent response) is composed of genes that are regulated by both Myc and ChREBP (Table S8). The elimination of either one, or both, leads to altered expression (either up or down). Group 2 (ChREBP-dependent response) is composed of genes regulated only by ChREBP and whose expression is not altered by Myc-KO. Group 3 (Myc-dependent response) is composed of genes only regulated by Myc and whose expression is not affected by ChREBP-KO. Group 4 (Conditional ChREBP response) is composed of genes whose expression is not altered by ChREBP binding; rather, ChREBP binding precludes Myc binding at the same or a different site. In ChREBP's absence, Myc now binds and alters expression. In the Double-KO state, a WT level of expression is restored, potentially through a third factor (X), which can only bind and/or activate its cognate gene in the absence of both Myc and ChREBP. This factor may or may not bind to E-box or ChoRE elements. Group 5 (Conditional Myc response) is related to the Group 4 response and is composed of genes that, in WT cells, are bound but not directly regulated by Myc. Rather, Myc insulates the gene from ChREBP, which, when bound, alters expression. As with Group 4, the restoration of normal target gene expression may be mediated via a third factor. Group 6 (Shared dependent response) is related to the Group 1 response and is composed of genes regulated by both Myc and ChREBP, either one of which is sufficient to insulate the gene from a third factor, which, when bound, restores a WT level of expression. Group 7 (Cooperative response) is composed of genes that may be bound by Myc and/or ChREBP but with equivalent responses under either condition. Only when both factors are absent is there a change in expression. C, flowchart representing the types of gene expression changes occurring in response to the knockout of Myc, ChREBP, or both, according to the terminology used in A and B. D, summary of the number of significant gene expression changes observed in ChREBP-KO, Myc-KO, and Double-KO livers when compared with WT liver, according to the Venn diagram layout shown in A (q < 0.05). E, summary of shared gene expression changes observed in tumors (q < 0.05). F, description of types of gene expression changes observed in ChREBP-KO, Myc-KO, and Double-KO tumors. Genes falling into each of the Venn diagram groups shown in A were analyzed using gene ontology and pathway analysis. Numbers in the top left corner of each box correspond to each of the seven groups. Up (red) and down (green) arrows signify general up- or down-regulation of genes belonging to a given pathway. Pathways without arrows contain dysregulated genes but could not be assigned directionality with any degree of confidence. An analysis for livers is not shown, as the number of dysregulated genes in each Venn diagram group were generally too low to reliably predict pathway directionality.
Figure 5.
Figure 5.
RPT expression in Myc-KO, ChREBP-KO, and Double-KO livers and tumors. A, average expression of all 80 RPTs across all experimental groups. p values were calculated using t tests corrected for multiple comparisons. B, raw expression (FPKM) of all 80 RPTs across all experimental groups displayed as a heat map. The order of transcripts is based on the most to the least abundant in WT livers (WL). C, PCA of RPT relative expression in livers and tumors. Livers and tumors cluster distinctly from one another, indicating that each group possesses distinct patterns of RPT expression. D, PCA of RPT expression performed separately in livers and tumors. WT livers cluster distinctly from knockout livers, but none of the knockout liver groups can be distinguished from one another. All tumor groups cluster distinctly from one another, however, indicating that knockout of Myc, ChREBP, or both leads to distinct expression patterns of RPTs.
Figure 6.
Figure 6.
Metabolic map of transcriptional changes in ChREBP-KO, Myc-KO, and Double-KO livers. Genes involved in direct metabolic processing of carbohydrates and lipids were mapped and grouped into general categories. Metabolic categories likely to be down-regulated relative to WT liver, either by significant down-regulation of genes encoding key rate-limiting enzymes or significant down-regulation of a large portion of many genes in the category, are colored green. Metabolic categories likely to be up-regulated in an experimental group relative to WT liver are colored red. A, summary of transcriptional changes in metabolic pathways in ChREBP-KO livers compared with WT livers, as described above. B, transcriptional changes in metabolic pathways in Myc-KO livers compared with WT livers. C, transcriptional changes in metabolic pathways in Double-KO livers compared with WT livers.
Figure 7.
Figure 7.
Metabolic map of transcriptional changes in ChREBP-KO, Myc-KO, and Double-KO tumors. A, summary of transcriptional changes in metabolic pathways in ChREBP-KO tumors compared with WT tumors, as described in Fig. 6. B, transcriptional changes in metabolic pathways in Myc-KO tumors compared with WT tumors. C, transcriptional changes in metabolic pathways in Double-KO tumors compared with WT tumors.

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