p27 is a key regulator of cell proliferation through inhibition of G 1 cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5 cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5 untranslated region (5-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.

p27Kip1 levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27Kip1 levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27 Kip1 enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27Kip1 . Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27 Kip1 involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Pur␣ to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed.Oligodendrocytes are the myelin-producing cells in the central nervous system. Differentiation of the progenitor cells into mature myelinating cells involves the activation of a genetic program that leads to expression of a set of genes encoding proteins important for the elaboration of the myelin membrane. The expression of these genes is at least partially regulated by transcription factor activity. These regulators of transcription include activators, repressors, and their cofactors. Transcriptional regulation may also involve chromatin modifications.One of the genes that is activated during differentiation of oligodendrocytes is the gene encoding myelin basic protein (MBP).1 This gene is exclusively expressed in mature oligodendrocytes, and its expression is mainly regulated at the transcriptional level. It provides an ideal model to investigate the mechanism of transcriptional regulation during cellular differentiation. Myelination begins in the mouse at ϳ9 days after birth, peaks at about postnatal day 17, and declines to a steady state by 2 months of age. MBP is encoded by a single gene in the mouse and human. The sequence of the MBP promoter from mouse, rat, and human has been determined, and it is highly conserved, especially in the core promoter region (1). In mouse brain, MBP mRNA is detected in oligodendrocytes at the end of the first postnatal week, peaks at 18 days, and remains at low levels in the adult (2). Thus expression of MBP is temporally regulated and is tightly correlated with myelination in the developing central nervous system.Previous work from our laboratory and the laboratories of others has shown that several different transcription factors are involved in activation of MBP expression in differen...

Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons. In the central nervous system, MBP is synthesized by differentiating oligodendrocytes. The expression of MBP in oligodendrocytes is regulated mainly at the transcriptional level. The Sp1 family of transcription factors has been shown to be important in the regulation of many genes. Binding of Sp1 to the GC box in the proximal MBP promoter has been shown to be indispensable for the activation of MBP gene expression. Previous results from our laboratory have shown that the increase in p27Kip1 that accompanies oligodendrocyte differentiation is paralleled by an increase in Sp1. We also have shown that the increase in MBP expression resulting from elevated p27Kip1 levels is mediated through Sp1 and that this effect occurs specifically in oligodendrocytes. In this study, we found that increased expression of p27Kip1 together with the nervous-system-specific transcription factor Sox10 can activate the MBP promoter even in nonoligodendrocyte cells. This indicates that Sox10 confers cell type specificity on the expression of MBP. Both Sp1 and Sox10 can enhance MBP promoter activity when expressed alone. Cotransfection of plasmids encoding Sp1 and Sox10 induces increased activation of the MBP promoter over expression of either transcription factor alone. This effect is not limited to oligodendrocyte cell lines, in that Sp1 and Sox10 can also synergistically activate the MBP promoter when expressed in NIH3T3 fibroblasts. Mutation of the Sp1 binding sites in the MBP promoter eliminates Sox10 stimulated activation, suggesting that the MBP promoter is activated, at least in part, through protein-protein interactions between Sp1 and Sox10.

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Using stable cell lines containing a series of deletions of the myelin basic protein (MBP) promoter directing the bacterial chloramphenicol acetyltransferase gene in a peripheral neurinoma cell line, we have studied the sequences in the MBP promoter needed for induction by cyclic AMP. Stimulation of expression from the MBP promoter by cyclic AMP is not a rapid response. Expression begins after 24 h and reaches a maximum at approximately 72 h. The results from the stable transformants indicate at least one region that appears to be essential to the induction of transcription directed by the MBP promoter. The region that is necessary for induction does not contain a consensus cyclic AMP response element. A specific binding site involved in the induction by cyclic AMP was localized to an NFI binding site.

The process of oligodendrocyte differentiation is a complex event that requires cell cycle withdrawal, followed by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. Because growth arrest precedes differentiation, we sought to investigate the role of cell cycle molecules in the activation of myelin gene promoters. We hypothesized that the cell cycle inhibitor p27(Kip1), which is primarily responsible for arresting proliferating oligodendrocyte progenitors, may be involved in the transcriptional regulation of myelin genes. In agreement with this hypothesis, overexpression of p27(Kip1) in the CG4 cell line, but not in 3T3 fibroblasts, enhances the expression of luciferase driven by the myelin basic protein (MBP) promoter. Interestingly, this effect is specific for p27(Kip1); overexpression of other cell cycle inhibitors had no effect. Additionally, this effect is independent of halting the cell cycle; treatment with the cell cycle blocker roscovitine did not affect MBP promoter usage. We conclude that p27(Kip1) contributes to oligodendrocyte differentiation by regulating transcription of the MBP gene.

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