Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

Direct observation of individual endogenous protein complexes in situ by proximity ligation

Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation.

SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.

Just over 25 years ago, MYC, the human homologue of a retroviral oncogene, was identified. Since that time, MYC research has been intense and the advances impressive. On reflection, it is astonishing how each incremental insight into MYC regulation and function has also had an impact on numerous biological disciplines, including our understanding of molecular oncogenesis in general. Here we chronicle the major advances in our understanding of MYC biology, and peer into the future of MYC research.

Reflecting on 25 years with MYC

Transcriptional Amplification in Tumor Cells with Elevated c-Myc

The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

The Myc/Max/Mad Network and the Transcriptional Control of Cell Behavior

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27 kip1 with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2–Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad–Max complexes and de‐repressed by Myc via a single highly conserved E‐box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase‐dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc‐dependent dissociation of p27 from Cdk2 and Myc‐induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2‐deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.

/pdf/direct-induction-of-cyclin-d2-by-myc-contributes-to-cell-4q4qorg6lv.pdf

Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.

Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. We now show that Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc.

/pdf/regulation-of-cyclin-d2-gene-expression-by-the-myc-max-mad-4yo5g91uim.pdf

Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter

https://www.cell.com/trends/cell-biology/pdf/S0962-8924(98)01251-3.pdf

Control of cell proliferation by Myc

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. We now show that Myc recruits TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. We suggest that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes.

Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins

Activation of Myc triggers a rapid induction of cyclin E/cdk2 kinase activity and degradation of p27. Overt degradation of p27 is preceded by a specific dissociation of p27 from cyclin E/cdk2, but not from cyclin D/cdk4 complexes. We now show that cyclin E/cdk2 phosphorylates p27 at a carboxy-terminal threonine residue (T187) in vitro; mutation of this residue to valine stabilises cyclin E/cdk2 complexes. This reaction is not significantly inhibited by high concentrations of p27, suggesting that cdk2 bound to p27 is catalytically active. In vivo, p27 bound to cyclins E and A, but not to D-type cyclins is phosphorylated. Myc-induced release of p27 from cdk2 requires cdk2 kinase activity and is delayed in a T187V mutant of p27. After induction of Myc, p27 phosphorylated at threonine 187 transiently accumulates in a non cdk2 bound form. Our data suggest a mechanism in which p27 is released from cyclin E/cdk2 upon phosphorylation; in Myc-transformed cells, release is efficient as phosphorylated p27 is transiently bound in a non-cdk2 containing complex and subsequently degraded.

Caroline Bouchard

Papers

Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.

Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter

Control of cell proliferation by Myc

Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins

Cdk2-dependent phosphorylation of p27 facilitates its Myc-induced release from cyclin E/cdk2 complexes