[Frontiers in Bioscience 3, d532-547, June 8, 1998]

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Amy S. Yee, Heather H. Shih, and Sergei G. Tevosian

The Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111

Received 5/10/98 Accepted 5/29/98


4.1. Working Model: "A Differentiation Checkpoint??"

Differentiation is a highly coordinated process of cell cycle exit with the expression of tissue specific genes. How do we rationalize the recent results addressing positive and negative regulation in differentiation? In the working model of figure 5, we hypothesize a "differentiation checkpoint" to insure orderly progression and fidelity in differentiation. This differentiation checkpoint model is a balance of positive and negative regulation to insure fidelity. Negative regulation (through p130, HBP1, CHOP, and/or p202) transiently suspends the differentiation pathway between cell cycle exit and tissue-specific gene expression. This would prevent inappropriate expression of tissue-specific genes until completion of cell cycle exit. The positive signals can now activate RB and subsequently MyoD (or other global regulators) to trigger the final activation of tissue-specific genes. A differentiation checkpoint involving RB family members provides tight coordination and temporal progression of cell cycle exit and tissue-specific gene expression.

Figure 5. Positive and Negative Regulation in Differentiation. The cell cycle exit and tissue-specific aspects of differentiation are summarized in this diagram. In the cell cycle exit phase, the coordinate actions of E2F4,5, p130 and HBP1 contribute to the repression of cell cycle genes. Additionally, RB and HBP1 may contribute to the irreversibility of cell cycle exit that is manifested in differentiation. We hypothesize a differentiation checkpoint consisting of positive and negative factors that insure precise coupling of cell cycle and tissue-specific regulation. RB contributes to positive activation of MyoD and other global regulators in an ill-defined mechanism. CHOP, HBP1, and p202 all contribute to negative regulation of MyoD-like factors, although each protein elicits cell cycle exit. Thus the ratio of positive and negative signals (denoted by RB/HBP) may be cellular "barometer" for the appropriate environment for differentiation. A low [RB]/[HBP1] ratio would favor cell cycle exit, but not tissue-specific gene expression. A high [RB]/[HBP1] ratio reflects an increase in RB and would favor the activation of MyoD-like factors and of tissue-specific gene expression.

The unexpected functional similarities of three distinct proteins (HBP1, CHOP, and p202) suggest the existence of a regulatory pathway that may insure that complete cell cycle exit. The completion then "signals" the positive activation of MyoD- or C/EBP-like transcription factors to trigger tissue-specific gene expression. The recent demonstration that p130 and/or p107 was inhibitory to differentiation may suggest a new function for RB family members. Additional RB targets with paradoxical functions in differentiation have been isolated (Kaelin, personal communication).

4.1.1. The Early Phase: Cell Cycle Exit and Apoptosis Protection

Figure 5 depicts a working model that integrates the recent observations on the relative functions of RB, p107, and p130 into the framework of a full differentiation pathway. We have divided differentiation into two general steps: cell cycle exit and tissue-specific gene expression. Considering existing data, E2F, HBP1, p130, RB collaborate to give general transcriptional repression of cell cycle genes. Whether HBP1 and p130 collaborate with E2F is currently unknown, but separately, each constitutes an efficient repression complexes. Regardless of the precise molecular interactions, the net effect is cell cycle exit. RB and HBP1 may also function in establishing the irreversibility of cell cycle arrest.

The initial phase of differentiation is characterized by extensive apoptosis, and RB may have some role in protection of apoptosis protection. Numerous studies in knockout mice and cellular systems now support a role for RB in apoptosis protection. While the specific mechanisms still require much work, a potential role for RB in the early phase of differentiation may be apoptosis protection, possibly with p21. The induction of p21 occurs in numerous differentiation models.

Figure 4 and 5 focus on the inhibition of the master regulators by HBP1 and p130 or RB. We speculate that two related mechanisms may work to insure fidelity and progression during differentiation. First, HBP1 and p130 may be an active inhibitor of MyoD and other master regulators of tissue-specific genes. Second, the relative ratio of overall HBP1 and RB concentrations may constitute a barometer for cell cycle exit or full differentiation. At low [RB] to [HBP1] ratios during the early phase of differentiation, cell cycle exit predominated, but tissue-specific gene expression was blocked. Future investigations must directly test these possibilities.

4.1.2. The Late Phase: Activation of Tissue-specific Gene Expression

The defining feature of differentiation is the expression of tissue-specific genes. The prevailing evidence suggests that RB has a positive role in the activation of tissue-specific genes in both fat and muscle. In either model, RB-/- cells fail to differentiate. Restoration of RB allows full differentiation through transcriptional activation by MyoD or c/EBP. The time course for the accumulation of the under-phosphorylated RB is also consistent with triggering MyoD and the activation of tissue-specific genes. Despite ubiquitous tissue expression, RB may be a necessary co-factor for activation of tissue-specific genes through MyoD and similar regulators. The simultaneous involvement of RB in cell cycle and tissue-specific regulation provides a convenient means to coordinate these processes in a full differentiation pathway. Additionally, a high overall [RB] to [HBP1] ratio in this phase may signal an appropriate environment for the final expression of tissue specific genes.

4.2. Future Perspectives

The past few years have provided new appreciation for the complexities in RB family function during cell differentiation. Numerous studies have established that cell cycle exit is clearly necessary for differentiation, since preventing exit by expression of oncogenes or growth factors block differentiation. While cell cycle exit is necessary, it is clearly not sufficient for full differentiation. Mechanisms must clearly exist to couple cell cycle exit with tissue-specific gene expression. Several recent studies now document proteins that elicit cell cycle exit, but also prevent differentiation. Recent studies on distinct functions of RB, p130 and p107 in differentiation also provide further evidence of a pathway that coordinates general and tissue-specific events. Collectively, we hypothesize a differentiation checkpoint that insures that appropriately arrested and viable cells can initiate tissue-specific gene expression.

Clearly, the studies of these novel coordination mechanisms are still in their infancy, but the early evidence does suggest an exciting, but complex view of differentiation. Based on the relative expression and on the tantalizing data from Classon and Harlow, a clear prediction is that p130 may have an inhibitory role in MyoD transcriptional activation. This putative inhibitory role for p130 must still be clearly demonstrated, but may be difficult with the existing functional redundancy in the RB family. Additionally, this model also predicts that complexes of HBP1 with RB or with p130 may have different functions through differentiation. For example, as RB levels increase, does RB displace p130 in a complex with HBP1? These questions require answers, but the low levels of HBP1 will surely hamper definitive results. Mice deficient in HBP1 and eventually crosses with mice deficient in RB, p107 and p130 may be needed to resolve these questions.

More work is necessary to establish the involvement of RB, p130, HBP1, and other proteins in coordinating cell cycle exit and tissue-specific gene expression in differentiation. The initial observations do suggest differential functions for RB, p107 and p130, and further experiments are necessary to solidify the differences. These important investigations will be difficult with the extensive functional redundancy. Furthermore, the precise mechanisms for positive activation of MyoD by RB are still unclear. If the [RB]/[HBP1] ratio is a "sensor" of differentiation, then how is this signal transduced into concrete molecular changes at the level of MyoD family transcriptional activation? Is there a direct phosphorylation event to alter transcriptional activation or DNA binding by MyoD? Or are there changes in MyoD’s physical interactions with p300 or MEF2c? Is RB "activated" to give overall enhanced MyoD transcriptional activation? If so, what are the signalling pathways?

For completion, we have documented apoptosis protection and the role of RB in differentiation. However, much work still remains in establishing how RB participate in general apoptosis protection during differentiation. An open question is the role of p21. To add complexity, a recent study in keratinocyte differentiation now suggest that p21 has functional similarities to HBP1, p202, and CHOP. While induced with differentiation and involved in cell cycle arrest, keratinocyte differentiation was blocked upon p21 expression (98). Again, cell cycle arrest and probably apoptosis protection by p21 was not sufficient to give keratinocyte differentiation.

An exciting future question will be how tissues acquire the fundamental features of cell cycle exit and apoptosis protection and trigger the necessary gene expression. We and others have provided only a glimpse of the complex positive and negative mechanisms that coordinate differentiation. Cell cycle exit must be a potent regulatory signal for the activation of MyoD and other factors to complete differentiation. An important future investigation will be the signal transduction pathways that intersect general cell cycle regulation with tissue-specific gene expression in differentiation.