A biochemical and biological analysis of Myc superfamily interactions.
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Amino Acid Sequence↗
Biomedical subjects
R A DePinho
Publications and source records attributed to R A DePinho.
Human CPR (cell cycle progression restoration) genes impart a Far- phenotype on yeast cells.
Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles.
Amino Acid Sequence↗
Role of the INK4a locus in tumor suppression and cell mortality.
The cell cycle inhibitor p16INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19ARF and the adjacent gene encoding p15I1NK4b, both negative regulators of cell proliferation. We report the phenotype of mice carrying a targeted deletion of the INK4a locus that eliminates both p16INK4a and p19ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate rapidly and have a high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-ras into INK4a-deficient fibroblasts can result in neoplastic transformation. These findings directly demonstrate that the INK4a locus functions to suppress neoplastic growth.
Alleles↗
Mouse Sin3A interacts with and can functionally substitute for the amino-terminal repression of the Myc antagonist Mxi1.
Mxi1 is a basic region helix-loop-helix leucine zipper (bHLH/LZ) protein that, in association with Max, antagonizes Myc oncogenic activities. A possible mechanistic basis for Mxi1-mediated repression was provided by the recent demonstration that the repressive potential of Mxi1 correlates with its ability to physically associate with mSin3B, one of two mammalian homologues of the yeast transcriptional repressor SIN3. Here, we sought to characterize more fully the physical properties of the second homologue, mSin3A and to determine whether the recruitment of mSin3A by Mxi1 is indeed required for anti-Myc activity. Transient transfection of mammalian cells showed that the mSin3A protein can associate with the strong repressive isoform of Mxi1 (Mxi1-SR) and that, like other Myc superfamily members, both mSin3A and Mxi1-SR localize to the nucleus. From a developmental standpoint, a comparative analysis of Myc, Mxi1-SR and Sin3A expression during postnatal mouse development and in differentiating mouse erythroleukemia (MEL) cells revealed that dramatic and reciprocal changes in Myc and Mxi1-SR mRNA levels are accompanied by minimal stage-specific changes in mSin3A gene expression. This constant expression profile, coupled with the observation that over-expression of mSin3A does not augment the anti-Myc activity of Mxi1-SR in the rat embryo fibroblast (REF) transformation assay, suggests that mSin3A is not a limiting factor in the regulation of Myc superfamily function. Finally, a mSin3A-Mxi1 fusion protein, in which the amino terminal mSin3-interacting domain of Mxi1-SR was replaced with the full-length mSin3A, exhibited a level of repression activity equivalent to, or greater than, the level of repression obtained with Mxi1-SR. Taken together, these observations directly demonstrate that the amino-terminal repression domain of Mxi1-SR functions solely to recruit mSin3A and possibly other proteins like mSin3A and this association is necessary for the anti-Myc activity of Mxi1-SR.
Amino Acid Sequence↗
Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.
A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.
Animals↗
Expression and activity of L-Myc in normal mouse development.
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.
Animals↗
Functional interactions among members of the Myc superfamily and potential relevance to cutaneous growth and development.
Myc family oncoproteins function as sequence-specific transcription factors that are believed to regulate the expression of genes governing cellular growth, differentiation, and programmed cell death. Activities of Myc are countered by those of Mad and Mxi1, two related members of the Myc superfamily. Mad and Mxi1 compete with Myc for common elements and interact with putative transcriptional repressors. While the precise role of the Myc superfamily in cutaneous biology remains to be determined, findings from a number of organ systems suggest that the regulated expression and function of its members are intimately correlated with proper development and physiology. Reviewed here are current data on Myc superfamily function with references where relevant to cutaneous processes with the ultimate goal of providing a framework upon which these proteins can be exploited in gene therapeutic approaches for diseases of the skin, including neoplasia.
Animals↗
Loss of transcriptional attenuation in N-myc is associated with progression towards a more malignant phenotype.
The modulation of RNA polymerase II processivity through the untranslated N-myc first exon represents an important mechanism governing N-myc mRNA levels during normal development. In this study, we employed the rat embryo fibroblast (REF) cooperation assay as a functional means to (i) map N-myc first exon sequences involved in the regulation of N-myc gene expression and specifically their contribution to controlling a block to transcriptional elongation and (ii) determine whether this transcriptional control mechanism plays a role in governing the oncogenic activity of N-myc. Transfection of N-myc expression constructs harboring various deletions within the untranslated first exon revealed that a region encoding a potential stem-loop structure followed by a thymine stretch (stem-loop/T region) was required for efficient transcriptional attenuation. The increase in transcriptional read-through associated with all deletions involving the stem-loop/T region also resulted in a more aggressive malignant phenotype as evidenced by a significant enhancement in the subcloning efficiency of N-myc/ras transformed foci. Most significantly, when cell lines generated from overexpression of the intact N-myc expression construct were selected for anchorage-independent growth, a strong block to transcriptional elongation was completely eliminated in all cases examined. Since the subcloning efficiency of transformed foci and the capacity of permanently established cell lines for anchorage-independent growth are direct correlates of more advanced stages of malignant transformation, our findings suggest that loss of transcriptional attenuation represents an important genetic event in the progression of N-myc-induced cellular transformation.
Animals↗
Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.
The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.
Animals↗
An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3.
Documented interactions among members of the Myc superfamily support a yin-yang model for the regulation of Myc-responsive genes in which transactivation-competent Myc-Max heterodimers are opposed by repressive Mxi1-Max or Mad-Max complexes. Analysis of mouse mxi1 has led to the identification of two mxi1 transcript forms possessing open reading frames that differ in their capacity to encode a short amino-terminal alpha-helical domain. The presence of this segment dramatically augments the suppressive potential of Mxi1 and allows for association with a mammalian protein that is structurally homologous to the yeast transcriptional repressor SIN3. These findings provide a mechanistic basis for the antagonistic actions of Mxi1 on Myc activity that appears to be mediated in part through the recruitment of a putative transcriptional repressor.
Amino Acid Sequence↗
Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo.
Although myc family genes are differentially expressed during development, their expression frequently overlaps, suggesting that they may serve both distinct and common biological functions. In addition, alterations in their expression occur at major developmental transitions in many cell lineages. For example, during mouse lens maturation, the growth arrest and differentiation of epithelial cells into lens fiber cells is associated with a decrease in L- and c-myc expression and a reciprocal rise in N-myc levels. To determine whether the down-regulation of L- and c-myc are required for mitotic arrest and/or completion of differentiation and whether these genes have distinct or similar activities in the same cell type, we have studied the consequences of forced L- and c-myc expression in the lens fiber cell compartment using the alpha A-crystallin promoter in transgenic mice (alpha A/L-myc and alpha A/c-myc mice). With respect to morphological and molecular differentiation, alpha A/L-myc lenses were characterized by a severely disorganized lens fiber cell compartment and a significant decrease in the expression of a late-stage differentiation marker (MIP26); in contrast, differentiation appeared to be unaffected in alpha A/c-myc mice. Furthermore, an analysis of proliferation indicated that while alpha A/L-myc fiber cells withdrew properly from the cell cycle, inappropriate cell cycle progression occurred in the lens fiber cell compartment of alpha A/c-myc mice. These observations indicate that continued late-stage expression of L-myc affected differentiation processes directly, rather than indirectly through deregulated growth control, whereas constitutive c-myc expression inhibited proliferative arrest, but did not appear to disturb differentiation. As a direct corollary, our data indicate that L-Myc and c-Myc are involved in distinct physiological processes in the same cell type.
Amino Acid Sequence↗
Inhibition of ras-induced proliferation and cellular transformation by p16INK4.
The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocked entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block was relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppressed cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth.
Adenovirus E1A Proteins↗
Effects of the MYC oncogene antagonist, MAD, on proliferation, cell cycling and the malignant phenotype of human brain tumour cells.
To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.
Animals↗
p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens.
The retinoblastoma tumour-suppressor gene (RB) has been implicated in negative growth regulation, induction of differentiation, and inhibition of cellular transformation. Homozygous inactivation of the Rb gene in the mouse leads to mid-gestational lethality with defects in erythropoiesis and neurogenesis. Here we describe the effects of the Rb-deficient state on the development of the ocular lens. The regional compartmentalization of growth, differentiation and apoptosis in the developing lens provides an ideal system to examine more closely the relationships of these processes in vivo. We demonstrate that loss of Rb function is associated with unchecked proliferation, impaired expression of differentiation markers, and inappropriate apoptosis in lens fibre cells. In addition, we show that ectopic apoptosis in Rb-deficient lenses is dependent on p53, because embryos doubly null for Rb and p53 show a nearly complete suppression of this effect. This developmental system provides a framework for understanding the consequences of the frequent mutation of both RB and p53 in human cancer.
Animals↗
Suppression of Myc, but not E1a, transformation activity by Max-associated proteins, Mad and Mxi1.
Mad and Mxi1, two members of the Myc-related basic-region helix-loop-helix/leucine-zipper family of proteins, associate directly with Max to form sequence-specific DNA binding heterodimers that are transactivation-incompetent. Mad-Max complexes have been shown to exert a strong repressive effect on Myc-induced transactivation, perhaps through the competitive occupation of common promoter binding sites also recognized by active Myc-Max heterodimers. To place these recent biochemical observations in a biological context, mad and mxi1 expression vectors were tested for their ability to influence Myc transformation activity in the rat embryo fibroblast cooperation assay. Addition of an equimolar amount of mad or mxi1 expression vector to mouse c-myc/ras cotransfections resulted in a dramatic reduction in both the number of foci generated and the severity of the malignant phenotype. Myc-specific suppression by Mad and Mxi1 was demonstrated by their ability to affect c- and N-myc-, but not ela-, induced transformation. In contrast, mad and mxi1 expression constructs bearing deletions in the basic region exerted only mild repressive effects on Myc transformation activity, suggesting that occupation of common DNA binding sites by transactivation-incompetent Mad-Max or Mxi1-Max complexes appears to play a more dominant role in this suppression than titration of limited intracellular pools of Max away from active Myc-Max complexes. Thus, these biological data support a current model for regulation of Myc function in which relative intracellular levels of Mad and Mxi1 in comparison to those of Myc may determine the degree of activation of Myc-responsive growth pathways.
Adenovirus E1A Proteins↗
Tumorigenic and developmental effects of combined germ-line mutations in Rb and p53.
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Animals↗
Evolutionary relationships and functional conservation among vertebrate Max-associated proteins: the zebra fish homolog of Mxi1.
In mammals, current evidence supports the view that Myc-responsive activities are regulated in part through an intracellular balance between levels of transcriptionally-active Myc/Max heterodimers and those of transcriptionally-inert Max/Max, Mad/Max and Mxi1/Max complexes. To gain insight into the roles of Mad and Mxi1 in cellular growth and differentiation and to fortify key structure-function relationships from an evolutionary standpoint, low stringency hybridization screens were used to identify potential homologs of these Max-associated proteins in the zebra fish genome. A single class of cDNA clones that cross-hybridized both to human mad and mxi1 probes was shown to encode a putative protein with significantly greater homology to mammalian Mxi1 than to Mad, particularly in the basic and helix-loop-helix (bHLH) regions. The high degree of structural relatedness between vertebrate Mxi1 proteins apparent in molecular modelling studies was consistent with the findings that the HLH/leucine zipper (LZ) region of zMxi1 exhibited the same profile of dimerization specificities as its mammalian counterpart in the two-hybrid system and that zmxi1 could, like human mxi1 (Lahoz et al., 1994), suppress the oncogenic potential of mouse c-myc in a mammalian cell. Finally, a comparison of steady-state zc-myc and zmxi1 mRNA levels during zebra fish embryogenesis demonstrated (i) high levels of zc-myc relative to zmxi1 mRNA during initiation of organogenesis, a period characterized by intense growth and active differentiation and (ii) rising levels of zmxi1 mRNA during progression towards the terminally differentiated state. These contrasting patterns of developmental expression together with the capacity of zmxi1 to repress myc-induced transformation support a model for the regulation, by Max-associated proteins, of Myc functions in the control of normal cell development and neoplastic growth.
Amino Acid Sequence↗
Use of transgenic mice to study myc family gene function in normal mammalian development and in cancer.
The myc family of cellular oncogenes (c-, N- and L-myc) encode for nuclear phosphoproteins that appear to regulate cellular proliferation and differentiation during normal vertebrate development. In addition, their overexpression has been correlated with malignant transformation and apoptotic cell death. The transgenic mouse technology has been utilized to study the unique and overlapping functions of the myc family in these cellular processes in the context of the developing animal. These studies suggest that Myc oncoproteins serve important roles during normal development and that their deregulated expression is causal in the development of many malignancies. Furthermore, these experiments have provided insight into the cell type specific activities of each gene and into the unique and overlapping roles of the genes during normal development.
Animals↗