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1.  Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice 
PLoS ONE  2010;5(9):e12739.
Background
Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Metfl/fl;Alb-Cre+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.
Methodology/Principal Finding
The priming events appeared to be intact in Metfl/fl;Alb-Cre+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Metfl/fl;Alb-Cre+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.
Conclusion/Significance
In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.
doi:10.1371/journal.pone.0012739
PMCID: PMC2940888  PMID: 20862286
2.  c-Myc affects mRNA translation, cell proliferation and progenitor cell function in the mammary gland 
BMC Biology  2009;7:63.
Background
The oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach.
Results
Generation of c-mycfl/fl mice carrying the mammary gland-specific WAPiCre transgene resulted in c-Myc loss in alveolar epithelial cells starting in mid-pregnancy. Three major phenotypes were observed in glands of mutant mice. First, c-Myc-deficient alveolar cells had a slower proliferative response at the start of pregnancy, causing a delay but not a block of alveolar development. Second, while milk composition was comparable between wild type and mutant animals, milk production was reduced in mutant glands, leading to slower pup weight-gain. Electron microscopy and polysome fractionation revealed a general decrease in translational efficiency. Furthermore, analysis of mRNA distribution along the polysome gradient demonstrated that this effect was specific for mRNAs whose protein products are involved in milk synthesis. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed decreased levels of ribosomal RNAs and ribosomal protein-encoding mRNAs in mutant glands. Third, using the mammary transplantation technique to functionally identify alveolar progenitor cells, we observed that the mutant epithelium has a reduced ability to repopulate the gland when transplanted into NOD/SCID recipients.
Conclusion
We have demonstrated that c-Myc plays multiple roles in the mouse mammary gland during pregnancy and lactation. c-Myc loss delayed, but did not block proliferation and differentiation in pregnancy. During lactation, lower levels of ribosomal RNAs and proteins were present and translation was generally decreased in mutant glands. Finally, the transplantation studies suggest a role for c-Myc in progenitor cell proliferation and/or survival.
See related minireview by Evan et al:
doi:10.1186/1741-7007-7-63
PMCID: PMC2761394  PMID: 19785743
3.  Mnt–Max to Myc–Max complex switching regulates cell cycle entry 
The Journal of Cell Biology  2005;169(3):405-413.
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt–Max complexes and a transient switch in the ratio of Mnt–Max to c-Myc–Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt–Max to c-Myc–Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.
doi:10.1083/jcb.200411013
PMCID: PMC2171929  PMID: 15866886
4.  Correct Timing of Proliferation and Differentiation is Necessary for Normal Inner Ear Development and Auditory Hair Cell Viability 
Background
Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1.
Results
Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation results in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule.
Conclusions
These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development.
doi:10.1002/dvdy.23910
PMCID: PMC3553268  PMID: 23193000
L-Myc; N-Myc; Hair Cell; Hearing; Prevention; Regeneration
5.  Post-translational control of Myc function during differentiation 
Cell Cycle  2011;10(4):604-610.
Myc proteins are deeply involved in multiple biological processes including cell proliferation, growth, metabolism, apoptosis, differentiation and tumorigenesis. Paradoxically, Myc proteins have been found to be capable of both inhibiting and facilitating differentiation depending on the biological context. Recently we identified a new mode of Myc regulation in differentiating muscle cells in which c-Myc protein is proteolytically cleaved by calcium-dependent calpains in the cytoplasm. This cleavage serves two purposes. First, it inactivates the transcriptional function of Myc by removing its C-terminus, a region responsible for the interaction of Myc with Max and DNA . Second, it alters cytoskeletal architecture and accelerates muscle differentiation through the activity of the remaining N-terminal cleavage product (termed Myc-nick). Here we discuss the roles and regulation of full-length Myc and Myc-nick in terminal differentiation and propose a model in which calpain-mediated cleavage of Myc operates as a functional switch.
doi:10.4161/cc.10.4.14794
PMCID: PMC3173997  PMID: 21293188
myc; differentiation; muscle; calpain; tubulin acetylation; acetyltransferase; GCN5
6.  The Myc 3′ Wnt-Responsive Element Regulates Homeostasis and Regeneration in the Mouse Intestinal Tract 
Molecular and Cellular Biology  2012;32(19):3891-3902.
The Wnt/β-catenin signaling pathway controls cellular proliferation in the intestines. In response to Wnt, β-catenin transits into the nucleus and associates with members of the T-cell factor (TCF) family of transcription factors. β-Catenin/TCF complexes bind Wnt responsive DNA elements (WREs) to activate target gene expression. The c-MYC proto-oncogene (MYC) is a direct target of β-catenin/TCF complexes. We recently identified the MYC 3′ WRE, which maps 1.4-kb downstream from the MYC transcription stop site. To investigate the role of the Myc 3′ WRE in the intestines, we generated a mouse model with a germ line deletion of this element. The intestinal architecture was largely preserved in knockout mice; however, removal of the Myc 3′ WRE compromised the crypt microenvironment. In comparison to wild-type intestines, knockout intestines contained an increased number of proliferative cells and a reduced number of differentiated cells comprising both absorptive and secretory lineages. Using a model of colitis, we found that knockout colons repaired more rapidly during the recovery period of the protocol. These results indicate that regulation of MYC expression through the Myc 3′ WRE contributes to intestinal homeostasis. Furthermore, our study implicates MYC as an important regulator of intestinal regeneration following injury.
doi:10.1128/MCB.00548-12
PMCID: PMC3457533  PMID: 22826434
7.  Myc Stimulates Nuclearly Encoded Mitochondrial Genes and Mitochondrial Biogenesis†  
Molecular and Cellular Biology  2005;25(14):6225-6234.
Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.
doi:10.1128/MCB.25.14.6225-6234.2005
PMCID: PMC1168798  PMID: 15988031
8.  c-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium 
Molecular and Cellular Biology  2005;25(17):7868-7878.
In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-mycflox allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreERT2 recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.
doi:10.1128/MCB.25.17.7868-7878.2005
PMCID: PMC1190312  PMID: 16107730
9.  c-myc Regulates Cell Proliferation during Lens Development 
PLoS ONE  2014;9(2):e87182.
Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc) is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27Kip1 proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens.
doi:10.1371/journal.pone.0087182
PMCID: PMC3913586  PMID: 24503550
10.  Hematopoietic Stem Cell Function and Survival Depend on c-Myc and N-Myc Activity 
Cell stem cell  2008;3(6):611-624.
SUMMARY
Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule GranzymeB, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.
doi:10.1016/j.stem.2008.09.005
PMCID: PMC2635113  PMID: 19041778
11.  Hepatotoxin-Induced Changes in the Adult Murine Liver Promote MYC-Induced Tumorigenesis 
PLoS ONE  2008;3(6):e2493.
Background
Overexpression of the human c-MYC (MYC) oncogene is one of the most frequently implicated events in the pathogenesis of hepatocellular carcinoma (HCC). Previously, we have shown in a conditional transgenic mouse model that MYC overexpression is restrained from inducing mitotic cellular division and tumorigenesis in the adult liver; whereas, in marked contrast, MYC induces robust proliferation associated with the very rapid onset of tumorigenesis in embryonic and neonatal mice.
Methodology/Principal Findings
Here, we show that non-genotoxic hepatotoxins induce changes in the liver cellular context associated with increased cellular proliferation and enhanced tumorigenesis. Both 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride (CCl4) cooperate with MYC to greatly accelerate the onset of liver cancer in an adult host to less than 7 days versus a mean latency of onset of over 35 weeks for MYC alone. These hepatotoxin-enhanced liver tumors grossly and histologically resemble embryonic and neonatal liver tumors. Importantly, we found that MYC overexpression is only capable of inducing expression of the mitotic Cyclin B1 in embryonic/neonatal hosts or adult hosts that were treated with either carcinogen.
Conclusion/Significance
Our results suggest a model whereby oncogenes can remain latently activated, but exposure of the adult liver to hepatotoxins that promote hepatocyte proliferation can rapidly uncover their malignant potential.
doi:10.1371/journal.pone.0002493
PMCID: PMC2423614  PMID: 18560566
12.  Developmental Context Determines Latency of MYC-Induced Tumorigenesis 
PLoS Biology  2004;2(11):e332.
One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.
A transgenic mouse model demonstrates that developmental context may be the reason why the spectrum of tumors differs in children and adults
doi:10.1371/journal.pbio.0020332
PMCID: PMC519000  PMID: 15455033
13.  Nucleophosmin is essential for c-Myc nucleolar localization and c-Myc-mediated rDNA transcription 
Oncogene  2012;32(15):10.1038/onc.2012.227.
The transcription factor c-Myc has a critical role in cell proliferation and growth. The control of ribosome biogenesis by c-Myc through the regulation of transcription mediated by all three RNA polymerases is essential for c-Myc-driven proliferation. Specifically, in the nucleolus, c-Myc has been shown to be recruited to ribosomal DNA and activate RNA polymerase (pol) I-mediated transcription of rRNA genes. In addition, c-Myc accumulates in nucleoli upon inhibition of the proteasome, suggesting nucleolar localization also has a role in c-Myc proteolysis. Nucleophosmin, a predominantly nucleolar protein, is also critical in ribosome biogenesis and, like c-Myc, is found overexpressed in many types of tumors. Previously we demonstrated that NPM directly interacts with c-Myc and controls c-Myc-induced hyperproliferation and transformation. Here, we show that NPM is necessary for the localization of c-Myc protein to nucleoli, whereas c-Myc nucleolar localization is independent of p53, Mdm2 and ARF. Conversely, high transient NPM expression enhances c-Myc nucleolar localization, leading to increased c-Myc proteolysis. In addition, NPM is necessary for the ability of c-Myc to induce rRNA synthesis in the nucleolus and constitutive NPM overexpression stimulates c-Myc-mediated rRNA synthesis. Taken together, these results demonstrate an essential role for NPM in c-Myc nucleolar localization and c-Myc-mediated rDNA transcription.
doi:10.1038/onc.2012.227
PMCID: PMC3855075  PMID: 22665062
c-Myc; NPM/B23; nucleolus; rRNA
14.  c-Myc/Max heterodimers bind cooperatively to the E-box sequences located in the first intron of the rat ornithine decarboxylase (ODC) gene 
Nucleic Acids Research  1997;25(8):1516-1525.
The oncoprotein c-Myc plays an important role in cell proliferation, transformation, inhibition of differentiation and apoptosis. These functions most likely result from the transcription factor activity of c-Myc. As a heterodimer with Max, the c-Myc protein binds to the E-box sequence (CACGTG), which is also recognized by USF dimers. In order to test differences in target gene recognition of c-Myc/Max, Max and USF dimers, we compared the DNA binding characteristics of these proteins in vitro using vaccinia viruses expressing full-length c-Myc and Max proteins. As expected, purified c-Myc/Max binds specifically to a consensus E-box. The optimal conditions for DNA binding by either c-Myc/Max, Max or USF dimers differ with respect to ionic strength and Mg2+ion concentration. Most interestingly, the c-Myc/Max complex binds with a high affinity to its natural target, the rat ODC gene, which contains two adjacent, consensus E-boxes. High affinity binding results from the ability of c-Myc/Max dimers to bind cooperatively to these E-boxes. We propose that differential cooperative binding by E-box binding transcription factors could contribute to target gene specificity.
PMCID: PMC146634  PMID: 9106360
15.  c-Myc/Max heterodimers bind cooperatively to the E-box sequences located in the first intron of the rat ornithine decarboxylase (ODC) gene. 
Nucleic Acids Research  1997;25(8):1493-1501.
The oncoprotein c-Myc plays an important role in cell proliferation, transformation, inhibition of differentiation and apoptosis. These functions most likely result from the transcription factor activity of c-Myc. As a heterodimer with Max, the c-Myc protein binds to the E-box sequence (CACGTG), which is also recognized by USF dimers. In order to test differences in target gene recognition of c-Myc/Max, Max and USF dimers, we compared the DNA binding characteristics of these proteins in vitro using vaccinia viruses expressing full-length c-Myc and Max proteins. As expected, purified c-Myc/max binds specifically to a consensus E-box. The optimal conditions for DNA binding by either c-Myc/Max, Max or USF dimers differ with respect to ionic strength and Mg2+ ion concentration. Most interestingly, the c-Myc/Max complex binds with a high affinity to its natural target, the rat ODC gene, which contains two adjacent, consensus E-boxes. High affinity binding results from teh ability of c-Myc/Max dimers to bind cooperatively to these E-boxes. We propose that differential cooperative binding by E-box binding transcription factors could contribute to target gene specificity.
PMCID: PMC146624  PMID: 9162900
16.  c-myc and N-myc promote active stem cell metabolism and cycling as architects of the developing brain 
Oncotarget  2010;1(2):120-130.
myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.
PMCID: PMC2907900  PMID: 20651942
c-myc; N-myc; brain tumors; stem cells; metabolism; mitosis; pluripotency
17.  c-myc and N-myc promote active stem cell metabolism and cycling as architects of the developing brain 
Oncotarget  2010;1(2):120-130.
myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.
PMCID: PMC2907900  PMID: 20651942
c-myc; N-myc; brain tumors; stem cells; metabolism; mitosis; pluripotency
18.  N-Myc and GCN5 Regulate Significantly Overlapping Transcriptional Programs in Neural Stem Cells 
PLoS ONE  2012;7(6):e39456.
Here we examine the functions of the Myc cofactor and histone acetyltransferase, GCN5/KAT2A, in neural stem and precursor cells (NSC) using a conditional knockout approach driven by nestin-cre. Mice with GCN5-deficient NSC exhibit a 25% reduction in brain mass with a microcephaly phenotype similar to that observed in nestin-cre driven knockouts of c- or N-myc. In addition, the loss of GCN5 inhibits precursor cell proliferation and reduces their populations in vivo, as does loss of N-myc. Gene expression analysis indicates that about one-sixth of genes whose expression is affected by loss of GCN5 are also affected in the same manner by loss of N-myc. These findings strongly support the notion that GCN5 protein is a key N-Myc transcriptional cofactor in NSC, but are also consistent with recruitment of GCN5 by other transcription factors and the use by N-Myc of other histone acetyltransferases. Putative N-Myc/GCN5 coregulated transcriptional pathways include cell metabolism, cell cycle, chromatin, and neuron projection morphogenesis genes. GCN5 is also required for maintenance of histone acetylation both at its putative specific target genes and at Myc targets. Thus, we have defined an important role for GCN5 in NSC and provided evidence that GCN5 is an important Myc transcriptional cofactor in vivo.
doi:10.1371/journal.pone.0039456
PMCID: PMC3383708  PMID: 22745758
19.  Cell density and paradoxical transcriptional properties of c-Myc and Max in cultured mouse fibroblasts. 
Journal of Clinical Investigation  1995;95(2):900-904.
Deregulated expression of the c-Myc oncoprotein occurs in several human malignancies. The c-Myc protein behaves as a transcription factor, and undoubtedly its role in carcinogenesis involves its ability to affect the expression of genes involved in cell growth. c-Myc has been reported to both activate and repress transcription in transient transfection experiments using reporter constructs bearing multiple copies of the c-Myc binding site, CAC (G/A) TG. We investigated these apparently paradoxical effects of c-Myc by determining if they arose from differences in the cell proliferation states of transfected cells. We found that endogenous c-Myc protein levels vary inversely with the degree of cell confluency, such that at low cell confluency, where endogenous levels of c-Myc are high and presumably endogenous levels of Max are limiting, exogenous c-Myc fails to affect basal transcription. In cells at high cell confluency, in which endogenous c-Myc levels are low, exogenous c-Myc augments transactivation by titrating the relative excess endogenous Max. These observations suggest that the apparently paradoxical behavior of c-Myc in transfection experiments is partially dependent on ambient cellular levels of c-Myc.
Images
PMCID: PMC295582  PMID: 7860774
20.  The opposing transcriptional functions of Sin3A and c-Myc are required to maintain tissue homeostasis 
Nature cell biology  2011;13(12):1395-1405.
How the proto-oncogene c-Myc balances the processes of stem cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc’s transcriptional roles at the Epidermal Differentiation Complex (EDC) a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovo1-1) and displace (C/EBPα, Mxi1 and Sin3A) specific sets of differentiation-specific transcriptional regulators to EDC genes. We found that Sin3A causes de-acetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3A, genomic recruitment of c-Myc to the EDC is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3A reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis via a negative feedback loop.
doi:10.1038/ncb2385
PMCID: PMC3242072  PMID: 22101514
21.  Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis 
PLoS Genetics  2012;8(3):e1002573.
c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, ApcMin mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes.
Author Summary
Cancer occurs when cells change their behavior and start to divide in an uncontrolled manner. To achieve this altered behavior, cells need to change their metabolism to be able to grow even when nutrient and oxygen supplies are limiting. Therefore, targeting metabolic pathways could be used to treat patients suffering from cancer. Here we studied a gene called MYC, which can regulate many metabolic pathways. By using genetically modified mice we can show that tumors have a remarkable ability to change their metabolism, even if key enzymes are removed. Taken together, our data suggest that metabolic disturbance by drugs in the clinic may present a future challenge.
doi:10.1371/journal.pgen.1002573
PMCID: PMC3305401  PMID: 22438825
22.  The C-Terminal of Nucleolin Promotes the Formation of the c-MYC G-Quadruplex and Inhibits c-MYC Promoter Activity† 
Biochemistry  2010;49(45):9706-9714.
Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85–90% of c-MYC’s transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroic spectropolarimetry (CD),1 thermal stability, and in vitro transcription. Here we report that nucleolin’s RNA binding domains (RBDs) 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription.
doi:10.1021/bi100509s
PMCID: PMC2976822  PMID: 20932061
23.  Stable transfection of CHO cells with the c-myc gene results in increased proliferation rates, reduces serum dependency, and induces anchorage independence 
Cytotechnology  2003;41(1):1-10.
Induction of the transcription factor Myc promotes cell proliferation and transformation by activating growth-promoting genes and/or by transcriptionally repressing the expression of growth arrest genes. However, a number of studies have shown that c-Myc is a potent inducer of apoptosis in the absence of serum or growth factors. To further examine the role of Myc in cell growth and proliferation, and the advantages of this positive regulator in cell culture we transfected the CHO-K1 cell line with a human c-myc gene driven by MMLV 5′-LTR promoter. Over-expression of ectopic c-Myc resulted in a significant increase in growth rate and maximum cell number, in both suspension and attached batch culture accompanied by a similar decrease in specific glucose consumption rate. Interestingly, there was no manifestation of the widely reported apoptotic death by c-myc in the absence of serum. Additionally, over-expression of c-Myc appeared to induce morphological transformation and partial anchorage-independence.
doi:10.1023/A:1024203518501
PMCID: PMC3449757  PMID: 19002957
Apoptosis; c-myc over-expression; CHO cells; Proliferation; Transformation
24.  Expression and activity of L-Myc in normal mouse development. 
Molecular and Cellular Biology  1996;16(4):1794-1804.
To determine the role of L-Myc in normal mammalian development and its functional relationship to other members of the Myc family, we determined the normal patterns of L-myc gene expression in the developing mouse by RNA in situ hybridization and assessed the phenotypic impact of L-Myc deficiency produced through standard gene targeting methodology. L-myc transcripts were detected in the developing kidney and lung as well as in both the proliferative and the differentiative zones of the brain and neural tube. Despite significant expression of L-myc in developing mouse tissue, homozygous null L-myc mice were found to be viable, reproductively competent, and represented in expected frequencies from heterozygous matings. A detailed histological survey of embryonic and adult tissues, characterization of an embryonic neuronal marker, and measurement of cellular proliferation in situ did not reveal any congenital abnormalities. The lack of an apparent phenotype associated with L-Myc deficiency indicates that L-Myc is dispensable for gross morphological development and argues against a unique role for L-Myc in early central nervous system development as had been previously suggested. Although overlapping expression patterns among myc family members raise the possibility of complementation of L-Myc deficiency by other Myc oncoproteins, compensatory changes in the levels of c- and/or N-myc transcripts were not detected in homozygous null L-myc mice.
PMCID: PMC231166  PMID: 8657155
25.  Transcriptional Repression 
Genes & Cancer  2010;1(6):580-586.
The (c-)Myc oncoprotein and its cousins, the N-Myc and L-Myc proteins, show all hallmarks of transcriptional activator proteins: Myc carries a carboxy-terminal DNA binding domain, which mediates sequence-specific binding to DNA. At its amino-terminus, Myc carries a transcriptional regulatory domain that strongly activates transcription when fused to an ectopic DNA binding domain; moreover, the strength of activation of different members of the Myc family correlates with their ability to transform rodent cells. Furthermore, activation of conditional alleles of Myc, either tetracycline or estrogen inducible, upregulates expression of a large number of genes, both in tissue culture and in transgenic animals. Indeed, many of these genes have essential roles in cell proliferation, cell growth, and metabolism; two of them, odc, encoding ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, and rpl24, encoding a constituent of the large ribosomal subunit, are haploinsufficient for Myc-induced lymphomagenesis but not for normal development, arguing very strongly that upregulation of both genes is critical for Myc-dependent tumor formation. Undoubtedly, therefore, Myc exerts part of its biological activities via transcriptional upregulation of a large number of target genes. One of the key issues in the field is whether there are additional biochemical activities of the Myc protein and, if so, whether and how they contribute to Myc biology. This review summarizes evidence demonstrating that Myc has the ability to repress transcription and that this may be an important function during oncogenic transformation.
doi:10.1177/1947601910379012
PMCID: PMC3092225  PMID: 21779459
Miz1; Sp1; p15Ink4b; p21Cip1; integrin beta1; Arf

Results 1-25 (601176)