The major transcription initiation site of the p27Kip1 gene is conserved in human and mouse and produces a long 5'-UTR.

BACKGROUND: The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different. RESULTS: In this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes. CONCLUSIONS: These results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1.

Control of cyclin-dependent kinase inhibitor p27 expression by cap-independent translation.

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.

A role for an AP-1-like site in the expression of the myelin basic protein gene during differentiation.

Differentiation of oligodendrocyte progenitors into mature oligodendrocytes involves the timely, cell-type specific expression of a number of different genes. Among these, the expression of the myelin basic protein (MBP) gene closely parallels the course of oligodendrocyte differentiation. To understand how transcription of the myelin basic protein gene is controlled, binding to the distal end of the 5' flanking sequence of the MBP gene was investigated. Specific protein-DNA complexes were localized to an AP-1-like element located between -1230 and -1240. The protein-DNA complexes formed at this site were shown to change as the cells differentiated. In undifferentiated cells two complexes were formed but, as the cells differentiated, binding was nearly completely lost. One of the two complexes was shown to contain a member of the fos family of transcription factors but no jun family members were involved. Mutation of the AP-1-like site resulted in loss of the complex and a change in expression of a reporter construct driven by the mutated promoter sequence. These results demonstrate a role for the AP-1-like site in repression of MBP gene expression in oligodendrocyte progenitor cells.

Mimosine arrests cells in G1 by enhancing the levels of p27(Kip1).

The plant amino acid mimosine, an effective inhibitor of DNA replication, has been demonstrated to arrest cell cycle progression in late G1. To understand further the molecular mechanism by which mimosine affects the cell cycle, we treated quiescent cells with serum in the presence of 800 microM mimosine. The cells did not enter S phase and were completely arrested in G1 phase. Although neither the mitogenic induction of the G1 cyclins nor the protein levels of cdk2 or cdk4 were affected, serum-dependent activation of cdk2 was blocked. This corresponded to elevated levels of the cdk inhibitor p27(Kip1). This was not mediated through inhibition of degradation but rather involved increased synthesis of both p27(Kip1) mRNA and protein. Mimosine did not appear to affect mitogen-dependent signals that normally lead to p27(Kip1) downregulation since the inhibitor was induced to even greater levels in quiescent, unstimulated cells. In the presence of mimosine, actinomycin D treatment for 2 h prevented the increase of p27(Kip1) mRNA, but p27(Kip1) protein levels were still enhanced under these conditions. We propose that mimosine blocks the cell cycle in late G1 phase by upregulation of p27(Kip1) protein levels through transcriptional and posttranscriptional regulatory mechanisms.

The cyclin-dependent kinase inhibitor p27Kip1 is localized to the cytosol in Swiss/3T3 cells.

p27Kip1 plays an important role in cell cycle progression by negatively regulating the activity of cyclin-Cdk complexes. To understand how p27Kip1 functions, the level and subcellular location of p27Kip1 in Swiss/3T3 cells following serum stimulation of quiescent cells was examined. Surprisingly, p27Kip1 was observed exclusively in the cytosol throughout G1 and into early S phase. However, as expected, p27Kip1 in the cytosolic fraction was greatly reduced following serum stimulation and reached very low levels by late G1. The decline in the level of p27Kip1 corresponded in time to an increase in the nuclear level of both Cdk2 and cyclin E. In quiescent 3T3 cells Cdk2 was inactive and co-precipitated with p27Kip1. After serum stimulation, both nuclear and cytosolic Cdk2 was activated and this corresponded to the decline in p27Kip1. Overexpression of p27Kip1 allowed accumulation of the inhibitor in the nucleus but inhibited entry of Cdk2 into the nucleus following serum stimulation. The subcellular localization of p27Kip1 was also examined in a variety of other mammalian cells. In all the cell lines examined the preponderance of p27Kip1 was found in the cytosolic fraction. However, a substantial level of nuclear p27Kip1 was observed for several cell lines. In a primary mixed glial cell culture p27Kip1 was localized to the nucleus. The results suggest that cytosolic p27Kip1 has a functional role in regulating cell cycle progression, possibly through inhibiting transport of cyclin E-Cdk 2 complexes into the nucleus.

ATF-1 is activated in response to UV irradiation in B16 melanoma cells.

We have found that the transferrin receptor gene promoter is strongly activated by exposure of B16 melanoma cells to UV light. This is a delayed event occurring more than 6 h after exposure and requires an AP-1/CRE-like element in the promoter as demonstrated by site-specific mutagenesis. UV irradiation enhances the binding of a nuclear factor to this element and supershift analysis demonstrates that this DNA-protein complex involves ATF-1. No other members of either the AP-1 or CREB/ATF families of transcription factors were found to bind to this DNA element in UV-irradiated B16 cells. Western blots show that the level of ATF-1 does not change following exposure to UV light, indicating that the increased binding of this factor is most likely mediated by posttranslational modifications in response to UV-mediated signaling pathways.

The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene.

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.

Involvement of MAP kinase in the cyclic AMP induction of myelin basic protein gene expression.

Cyclic AMP is involved in the differentiation of oligodendrocyte and Schwann cell progenitors into mature myelin producing cells. The involvement of MAP kinases in this pathway was investigated in the D6P2T cell line. This cell line can be induced to display a differentiated phenotype characterized by myelin basic protein gene expression by increased cyclic AMP. Blocking MAP kinase activity with inhibitors of the activating kinase, MEK, by expression of a dominant negative MAP kinase or by expression of the MAP kinase inactivating phosphatase Mkp-1 all blocked the activation of the myelin basic protein promoter in D6P2T cells. In addition, blocking MAP kinase activation during differentiation of an oligodendrocyte-like cell line, CG4, also leads to inhibition of MBP expression. These findings suggest a role for MAP kinase in the cyclic AMP stimulated expression of the myelin basic protein gene during differentiation.

Cyclin-dependent kinase inhibitor p27kip1 is expressed at high levels in cells that express a myelinating phenotype.

Terminal cellular differentiation is generally accompanied by exit from the cell cycle but the molecular basis of how the two events are coupled is poorly understood. In the central nervous system (CNS) the terminally differentiated, non-proliferating myelin-synthesizing cells, oligodendrocytes, arise from stem cells that are proliferation competent. To study the molecular mechanisms that link oligodendrocyte differentiation and cell cycle control, the D6P2T cell line has been used. This cell line responds similarly to oligodendrocytes in culture in response to increased cyclic AMP (cAMP). Upon increasing cAMP levels, D6P2T cells increase transcription of the endogenous myelin basic protein (MBP) gene. The increase in MBP gene transcription is accompanied by withdrawal of the cells from the cell cycle. The mechanism of cell cycle withdrawal in response to cAMP was found to involve a dramatic increase in the level of the cyclin-dependent kinase (cdk) inhibitor p27kip1 with little or no change in the levels of the cyclins D1 and E. The increase in p27kip1 is at least partially attributable to an increase in the mRNA levels for p27kip1. A striking increase in the cdk inhibitor p27kip1 was also shown to occur in vivo in oligodendrocytes, the cells responsible for myelination in the CNS. In contrast to D6P2T cells, however, this increase in p27kip1 was accompanied by a decrease in the levels of cyclin E.

Phosphatidylinositol 3-kinase inhibitor wortmannin blocks mitogenic activation of the transferrin receptor gene promoter in late G1.

Expression of the transferrin receptor is necessary for cells to progress through S-phase. The transferrin receptor gene promoter is activated as a delayed event following growth factor stimulation of quiescent fibroblasts. Serum stimulation in the presence of vanadate leads to superactivation of the transferrin receptor promoter, suggesting a role for tyrosine phosphorylation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, a tyrosine kinase-regulated enzyme, blocks mitogen-dependent activation of the transferrin receptor promoter. Furthermore, wortmannin was able to block activation of this promoter when added several hours after serum stimulation of quiescent cells. This suggests that phosphatidylinositol 3-kinase may be required in mid to late G1 and that it is directly involved in a pathway leading to activation of the transferrin receptor promoter. This is further supported by the finding that the transferrin receptor promoter is much less responsive to mitogenic stimulation in cells that have been stably transfected with a dominant negative form of the phosphatidylinositol 3-kinase regulatory subunit. Activation of S6 kinase, an event known to be downstream of phosphatidylinositol 3-kinase activation, appears not to be involved in activation of the transferrin receptor promoter since no effect was observed by treatment of cells with rapamycin.

Mitogenic activation of the transferrin receptor gene promoter is modulated by inhibitors of tyrosine kinases and tyrosine phosphatases.

Transferrin receptor gene expression is coupled to cell proliferation of normal cells and is elevated in nearly all types of tumor cells. A mitogen-responsive region of the transferrin receptor gene promoter has been localized between -78 and -34 relative to the major transcriptional start. The promoter can be activated in quiescent fibroblasts by treatment with either vanadate or phenylarsine oxide, both of which are inhibitors of tyrosine phosphatases and lead to elevated levels of intracellular tyrosine-phosphorylated proteins. Vanadate can act synergistically with other mitogens and, when added together with serum, leads to superactivation of the promoter. Genistein, an inhibitor of certain tyrosine kinases, actually enhances promoter activity in cells treated with either vanadate or phenylarsine oxide. On the other hand, geldanamycin, which also reduces the level of tyrosine-phosphorylated proteins and promoters the morphological reversion of many transformed cell types, is a potent inhibitor of transferrin receptor promoter activation by mitogens. The differential effects of these two tyrosine kinase inhibitors is most likely caused by the specificity of the enzymes that they target. These results indicate that a tyrosine phosphorylation event plays a critical role in the signaling events that lead to activation of the transferrin receptor gene promoter in mitogen-stimulated cells. This is of interest because activation of this promoter is a delayed response that occurs several hours after mitogen addition.

Involvement of protein kinase C in cAMP regulation of myelin basic protein gene expression.

Since synthesis of myelin components has been seen to be stimulated by cAMP in both oligodendrocytes and Schwann cells we have begun investigating the specific sequence(s) in the 5' flanking region of the myelin basic protein (MBP) gene that are responsible for the induction of MBP transcription by cAMP. Using stably transfected cell lines containing various deletions of the MBP promoter directing the bacterial chloramphenicol acetyltransferase (CAT) gene we have identified a region of the MBP gene that is inhibitory to stimulation by increased cAMP levels. This inhibition can be overcome by pretreating the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 48 hr. The effects on MBP gene expression modulated by TPA and cAMP involve altered DNA-protein interactions in the 5' end of the MBP promoter. The effect of TPA also appears to be mediated by down-regulation of protein kinase C.

Binding at an NFI site is modulated by cyclic AMP-dependent activation of myelin basic protein gene expression.

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.

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice.

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5' flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene down-stream of the MBP 5' flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5' flanking sequence is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

A theoretical model for the practice of residential treatment.

This paper presents a theoretical model describing the practice of psychiatric residential treatment for children and adolescents. The emphasis is on forty practice principles, guiding concepts which dictate the specific treatment techniques and administrative procedures for the Southern Oregon Adolescent Study and Treatment Center. These principles are grouped into six clusters, each a critical area of concern for residential treatment: program organization, physical environment, program personnel, clinical practices, therapeutic milieu, and interpersonal relationships.

Heterosis for myelin content is limited to the central nervous system.

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post-natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.

Molecular basis for heterosis for myelin basic protein content in mice.

Poly(A)+ mRNA was isolated from the brains of C57BL/6J (B6), DBA/2J (D2), and F1 hybrid mice (B6 X D2) of 16-17 days of age. The yield of polysomal RNA, both poly(A)+ and poly(A)-, from the three strains of mice was comparable. When translated in vitro in a reticulocyte lysate system, the mRNA preparations had the same efficiency with respect to stimulation of amino acid incorporation into protein. However, a significant heterotic effect was seen for the production of myelin basic protein (MBP) by the mRNA from the F1 mice. That is, the fraction of protein synthesized as MBP was greater for the F1 hybrid than for either parental strain. The distribution of the form of MBPs was not different among the three strains. We therefore believe that heterosis for brain MBP content in the F1 hybrid may be regulated at the transcriptional or post-transcriptional level.

Developmental expression of myelin basic protein mRNA in a hypermyelinating mouse.

Polysomal poly (A)+ RNA and total cellular RNA were isolated from the brains of C57BL/6J (B6), DBA/2J (D2), and F1 hybrid mice. Both types of RNA were isolated at 9, 12, 16, 21, and 30 days of age. By using a 32P-labelled cDNA probe for myelin basic protein (MBP; pMBP-1) the amounts of RNA coding for MBP present in the three strains at each age were determined. For both the polysomal poly(A)+ and total cellular RNA the peak of MBP RNA accumulation was at about 16-18 days for all three strains. These results imply that the developmental pattern of MBP RNA synthesis for F1 mice is not shifted with respect to the parental strains. For the polysomal poly(A)+ RNA, F1 had the greatest amount of MBP mRNA, B6 the least, and D2 an intermediate amount. F1 again had the greatest amount of MBP RNA from the total cellular pool at all ages. However, B6 had a greater amount of MBP RNA in the total cellular RNA pool than did D2. This demonstrates elevated transcription of the MBP gene in the F1 or an increase in message stability in this strain. It also suggests some form of translational control for MBP RNA in the B6 strain. In addition a second species of MBP RNA was found in the total cellular RNA pool. This species is larger than the major polysomal RNA band and may be a nuclear precursor.

Epidermal growth factor-induced topoisomerase(s). Intracellular translocation and relation to DNA synthesis.

We have used epidermal growth factor (EGF) to investigate the relationship between eukaryotic topoisomerases and DNA synthesis. We found that EGF stimulates topoisomerase activity in human fibroblasts and Swiss/3T3 mouse fibroblasts. The first increase is seen in the cytoplasm, followed by increased activity in the nucleus. The nuclear increases correspond to increases in DNA synthesis. A type II topoisomerase is stimulated as indicated by the ATP dependence of the relaxing reaction and by the formation of catenanes. We have also found that the topoisomerase activity in the cytoplasm is sedimentable indicating that it is either membrane-associated or in a supramolecular complex. The stimulation of topoisomerase activity by EGF may represent a key step in the process by which EGF induces DNA synthesis and cell division.