ABSTRACTcDNAs representing the a subunit of polyomavirus enhancer binding protein 2 (PEBP2; also cafled PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion 13 subunit. The human AMLI gene related to t(8;21) acute myeloid leukemia also has a run homology region. Together with the 18 subunit, which increases the affinity of the a subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2a mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is,involved in regulation of T-ceil-specific gene expression.Potential use of polyomavirus (Py) as a probe of mouse development has been recognized since it was observed that the wild-type Py did not grow in embryonal carcinoma cells but that it grew when the cells were induced to differentiate (1)(2)(3)(4)(5). Through the analysis of the Py enhancer, which determines the differentiation-dependent and cell-typespecific expression of the viral genes, a number of transcription factors involved in developmental regulation have been identified (6-10).PEBP2/PEA2, which binds to both the A and B cores of the Py enhancer (11), is undetectable in embryonal carcinoma F9 cells and becomes detectable after the cells are induced to differentiate (6,7). It specifically recognizes a consensus sequence, R/TACCRCA (R, purine), which was originally identified in the Py enhancer (11) and is also compatible with the core motif of murine leukemia virus enhancers (12,13). In addition, many T-cell-specific genes, such as T-cell receptor (TCR) a, X3, yand 8 and CD3e genes, contain potential PEBP2 binding sites, suggesting the possibility that PEBP2 is involved in T-cell-specific gene expression (13 [708][709][710][711][712][713][714]. The mutations are the same as the M2 mutation (14). For expression in mammalian cells, the coding regions of al, a2, and 831 cDNAs were cloned into the Xho I site of pCDMPy, in which the Py origin and enhancer region were deleted from pCDM8 (Invitrogen), resulting in pCDMPy-al, pCDMPy-a2, and pCDMPy-31. The bacterial expression plasmids pETal and pETa2, carrying the whole coding regions of al and a2 cDNAs, were constructed in the same way as pETf82 (16). To produce deletion mutants of a2, the whole coding region of a2 cDNA was inserted into the BamHI/HindIII fragment of plasmid pQE9 (Qiagen) resulting in pQE9-a2 encoding a fusion protein tagged with an N-terminal histidine cluster. Various deletions were introduced into pQE9-a2 using appropriate restriction sites: N94C306 encodes a region of aa 94-306; N1C226 encodes aa 1-226; N1C158 encodes aa 1-158; N80C226 encodes aa 80-226. N1C226, N1C158, and N80C226 proteins have one, two, o...

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 ,, or / 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 background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the / 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.

Edited by Eric FearonHepatoblastoma (HB) is associated with aberrant activation of the ␤-catenin and Hippo/YAP signaling pathways. Overexpression of mutant ␤-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc ؉/؉ (WT) and myc ؊/؊ (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid ␤-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid ␤-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetylCoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc−/− (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc−/− livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.