Retinoblastoma protein and MyoD function together to effect the repression of Fra-1 and in turn cyclin D1 during terminal cell cycle arrest associated with myogenesis.

The acquisition of skeletal muscle-specific function and terminal cell cycle arrest represent two important features of the myogenic differentiation program. These cellular processes are distinct and can be separated genetically. The lineage-specific transcription factor MyoD and the retinoblastoma protein pRb participate in both of these cellular events. Whether and how MyoD and pRb work together to effect terminal cell cycle arrest is uncertain. To address this question, we focused on cyclin D1, whose stable repression is required for terminal cell cycle arrest and execution of myogenesis. MyoD and pRb are both required for the repression of cyclin D1; their actions, however, were found not to be direct. Rather, they operate to regulate the immediate early gene Fra-1, a critical player in mitogen-dependent induction of cyclin D1. Two conserved MyoD-binding sites were identified in an intronic enhancer of Fra-1 and shown to be required for the stable repression of Fra-1 and, in turn, cyclin D1. Localization of MyoD alone to the intronic enhancer of Fra-1 in the absence of pRb was not sufficient to elicit a block to Fra-1 induction; pRb was also recruited to the intronic enhancer in a MyoD-dependent manner. These observations suggest that MyoD and pRb work together cooperatively at the level of the intronic enhancer of Fra-1 during terminal cell cycle arrest. This work reveals a previously unappreciated link between a lineage-specific transcription factor, a tumor suppressor, and a proto-oncogene in the control of an important facet of myogenic differentiation.

Cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Both cyclin D1 and the transcription factor C/EBPß are required for mammary epithelial cell differentiation; however, the pathway in which they operate is uncertain. Previous analyses of the patterns of gene expression in human tumors suggested a connection between cyclin D1 overexpression and C/EBPß, but whether this represents a cancer-specific gain of function for cyclin D1 is unknown. C/EBPß is an intronless gene encoding three protein isoforms--LAP1, LAP2, and LIP. Here, we provide evidence that cyclin D1 engages C/EBPß in an isoform-specific manner. Cyclin D1 binds to LAP1, an event that activates the transcriptional function of LAP1 by relieving its autoinhibited state effected by intramolecular interactions. Reexpression of LAP1 but not LAP2 or LIP restores the ability of C/EBPß-deficient mammary epithelial cells to differentiate and does so in a manner dependent on cyclin D1. And cyclin D1-mediated activation of LAP1 participates in mammary epithelial cell differentiation. Our findings indicate that cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Wild-type NRas and KRas perform distinct functions during transformation.

The ras proto-oncogenes, of which there are four isoforms, are molecular switches that function in signal transduction pathways to control cell differentiation, proliferation, and survival. How the Ras isoforms orchestrate cellular processes that affect behavior is poorly understood. Further, why cells express two or more Ras isoforms is unknown. Here, using a genetically defined system, we show that the presence of both wild-type KRas and NRas isoforms is required for transformation because they perform distinct nonoverlapping functions: wild-type NRas regulates adhesion, and KRas coordinates motility. Remarkably, we find that Ras isoforms achieve functional specificity by engaging different signaling pathways to affect the same cellular processes, thereby coordinating cellular outcome. Although we find that signaling from both isoforms intersects in actin and microtubule cytoskeletons, our results suggest that KRas signals through Akt and Cdc42 while NRas signals through Raf and RhoA. Our analyses suggest a previously unappreciated convergence of different Ras isoforms on the dynamics of the processes involved in transformation.

Effects of depletion of CREB-binding protein on c-Myc regulation and cell cycle G1-S transition.

We recently reported that the transcriptional coactivator and histone acetyltransferase p300 plays an important role in the G(1) phase of the cell cycle by negatively regulating c-myc and thereby preventing premature G(1) exit (Kolli, et al. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4646-4651; Baluchamy, et al. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 9524-9529). Because p300 does not substitute for all CREB-binding protein (CBP) functions, we investigated whether CBP also negatively regulates c-myc and prevents premature DNA synthesis. Here, we show that antisense-mediated depletion of CBP in serum-deprived human cells leads to induction of c-myc and that such cells emerge from quiescence without growth factors at a rate comparable with that of p300-depleted cells. The CBP-depleted cells contained significantly reduced levels of the cyclin-dependent kinase inhibitor p21 and low levels of p107 and p130 (but not pRb) phosphorylation, suggesting that these factors, along with elevated levels of c-Myc, contribute to induction of DNA synthesis. Antisense c-Myc reversed the phosphorylation of p107 and p130 and the induction of S phase in CBP-depleted cells, indicating that up-regulation of c-myc is directly responsible for the induction of S phase. Furthermore, the serum-stimulated p300/CBP-depleted cells did not traverse beyond S phase, and a significant number of these cells died of apoptosis, which was not related to p53 levels. These cells also contained significantly higher levels of c-Myc compared with normal cells. When c-myc expression was blocked by antisense c-Myc, the apoptosis of the serum-stimulated CBP-depleted cells was reversed, indicating that high levels of c-Myc contribute to apoptosis. Thus, despite their high degree of structural and functional similarities, normal levels of both p300 and CBP are essential for keeping c-myc in a repressed state in G(1) and thereby preventing inappropriate entry of cells into S phase. In addition, both these proteins also provide important functions in coordinated cell cycle progression.

Repression of c-Myc and inhibition of G1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity.

p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two highly homologous, conserved transcriptional coactivators, and histone acetyltransferases (HATs) that link chromatin remodeling with transcription. Cell transformation by viral oncogene products such as adenovirus E1A and SV40 large T antigen depends on their ability to inactivate p300 and CBP. To investigate the role of p300 in cell-cycle progression, we constructed stable rat cell lines, which conditionally overexpress p300 from a tetracycline-responsive promoter. When p300 was induced in these cells, serum-stimulated S-phase entry was significantly inhibited. The inhibition of S-phase induction was associated with down-regulation of c-Myc, but not of c-Fos or c-Jun. Simultaneous overexpression of c-Myc and p300 before serum stimulation reversed the inhibition of S-phase induction to a significant level, indicating that the inhibition of c-Myc to a large extent is responsible for the p300 inhibition of G1 exit. Similar studies with stable rat cell lines that overexpress a mutant p300, which lacks the HAT activity, showed that the intrinsic HAT activity of p300 is not required for the negative regulation of c-Myc or G1. These findings, and our previously published results (Kolli, S., Buchmann, A. M., Williams, J., Weitzman, S. & Thimmapaya, B. (2001) Proc. Natl. Acad. Sci. USA 98, 4646-4651), establish an important negative regulatory role for p300 in c-Myc expression that may be important in maintaining the cells in the G0/G1 phase of the cell cycle.