Identification and functional characterization of an intragenic DNA binding site for the spumaretroviral trans-activator in the human p57Kip2 gene.
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Expression of the human cyclin-dependent protein kinase inhibitor p57(Kip2) gene was previously shown to be specifically and strongly activated by the retroviral trans-activator Bel1 of human foamy virus by means of expression profiling, Northern, and Western blot analysis. Here we report that Bel1-mediated trans-activation was conferred by a Bel1 response element (BRE) located in the second exon of p57(Kip2). The intragenic Kip2-BRE was capable of trans-activating the luciferase reporter gene upon cotransfection with Bel1. In electrophoretic mobility shift assays using 293T nuclear extracts or a purified glutathione S-transferase (GST) small middle dotBel1 fusion protein, we identified the 55-nucleotide-long Kip2-BRE site that mainly consists of three direct repeats of 14-mers partially homologous to a functionally active BRE in the viral internal promoter. The specificity of the transactivator-DNA binding was shown by using mutated and shortened Kip2-BRE oligodeoxynucleotides in competition experiments with the authentic viral internal promoter and by Bel1-specific antibody that led to a supershift of the nuclear protein small middle dotKip2-BRE and GST small middle dotBel1 small middle dotKip2-BRE complex. The data indicate that Bel1 can directly bind to BRE sites. The cellular Kip2-BRE can be used to predict those human genes that are directly or indirectly activated by the Bel1 trans-activator. (+info)
Pituitary adenylate cyclase activating polypeptide anti-mitogenic signaling in cerebral cortical progenitors is regulated by p57Kip2-dependent CDK2 activity.
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Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57Kip2-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27Kip1, p57Kip2) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G1/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57Kip2 protein, but not p21Cip1 or p27Kip1, suggesting that p57Kip2 mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57Kip2 association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57Kip2 interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex. (+info)
Induction of p57(KIP2) expression by p73beta.
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The p53-related protein p73 has many functions similar to that of p53 including the ability to induce cell-cycle arrest and apoptosis. Both p53 and p73 function as transcription factors, and p73 activates expression of many genes that also are regulated by p53. Despite their similarities, it is evident that p53 and p73 are not interchangeable functionally, with p73 playing a role in normal growth and development that is not shared by p53. In this paper we describe the ability of p73beta but not p53 to activate expression of the cyclin-dependent kinase inhibitor p57(KIP) and KvLQT1, two genes that are coregulated in an imprinted region of the genome. Our results suggest that p73 may regulate expression of genes through mechanisms that are not shared by p53, potentially explaining the different contributions of p53 and p73 to normal development. (+info)
Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors.
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To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5' CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P<0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (-300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors. (+info)
Renal abnormalities in beckwith-wiedemann syndrome are associated with 11p15.5 uniparental disomy.
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Beckwith-Wiedemann syndrome (BWS) is a somatic overgrowth syndrome characterized by a variable incidence of congenital anomalies, including hemihyperplasia and renal malformations. BWS is associated with disruption of genomic imprinting and/or mutations in one or more genes encoded on 11p15.5, including CDKN1C (p57(KIP2)). It was hypothesized that genotypic and epigenotypic abnormalities of the 11p15.5 region affecting CDKN1C were associated with renal abnormalities. Medical records for 159 individuals with BWS were reviewed. All underwent at least one abdominal ultrasonographic evaluation. Testing for paternal uniparental disomy (UPD) at 11p15.5, CDKN1C mutations, and imprinting defects at KvDMR1 was performed for 96, 32, and 47 patients, respectively. Of the 159 patients, 67 (42%) exhibited renal abnormalities, mainly nephromegaly (25%), collecting system abnormalities (11%), and renal cysts (10.5%). The frequency of renal lesions among patients who were tested for genetic abnormalities did not differ from that among patients who were not tested. Paternal UPD was demonstrated in 22 of 96 cases (23%), CDKN1C mutations in eight of 32 cases (25%), and KvDMR1 imprinting defects in 21 of 47 cases (45%). The 22 UPD-positive patients exhibited a significantly higher incidence of renal abnormalities (P = 0.0026). Surprisingly, the eight patients with CDKN1C mutations exhibited no significant increase in the incidence of renal lesions (P = 0.29). Imprinting defects at KvDMR1, which might downregulate CDKN1C, were also not associated with a significant difference in the incidence of renal disease. Whereas UPD at 11p15.5 in BWS was associated with a higher incidence of renal abnormalities, mutations at CDKN1C and KvDMR1 imprinting defects were not, suggesting that imprinted genes on 11p15.5 other than CDKN1C are critical for renal development. (+info)
Mammalian achaete scute homolog 2 is expressed in the adult sciatic nerve and regulates the expression of Krox24, Mob-1, CXCR4, and p57kip2 in Schwann cells.
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The molecular control mechanisms and regulatory molecules involved in nerve repair are not yet well known. Schwann cells have been attributed an important role in peripheral nerve regeneration; therefore, attention has been drawn to regulatory factors expressed by these glial cells. Here, we demonstrate that Mash2, a basic helix-loop-helix (bHLH) transcription factor previously shown to be crucial for placenta development, is expressed by Schwann cells of adult peripheral nerves. We observed that this gene is downregulated after nerve lesion and, using cDNA array hybridization technology, we could demonstrate that Mash2 is a regulator of Krox24, Mob-1, and CXCR4 expression in cultured Schwann cells. In addition, we provide strong evidence that Mash2 is a negative regulator of Schwann cell proliferation. Mash2 represents a first candidate for the missing class B bHLH proteins in peripheral nerves. (+info)
Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation. (+info)
Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases.
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As in many cell types, lens cells must withdraw from the cell cycle before they initiate their differentiation. The involvement of Src family kinases (SFKs) in this key initiating event in cell differentiation was examined in lens epithelial cell cultures. SFK activity was suppressed with the specific inhibitor PP1. This induced expression of the cyclin-dependent kinase (CDK) inhibitors p27 and p57 and suppressed lens epithelial cell proliferation. Therefore, inhibition of SFK activity created conditions permissive for undifferentiated lens epithelial cells to withdraw from the cell cycle. Growth of the lens epithelial cell cultures in the presence of PP1 induced expression of filensin and CP49, lens differentiation-specific intermediate filament proteins, providing evidence that suppression of SFK activity also promoted the initiation of lens cell differentiation. The mechanism by which PP1 signaled cell cycle withdrawal and commitment to differentiation was shown to involve induction of N-cadherin cell-cell junction assembly and reorganization of the actin cytoskeleton from stress fibers to cortical filaments. This result was supported by the compaction of the epithelial monolayer in response to PP1, a morphogenetic change that we have previously shown to be dependent on N-cadherin function and a hallmark of the commencement of the lens differentiation program in culture. The results presented in this study suggest that the decision of lens epithelial cells to withdraw from the cell cycle and initiate differentiation requires inhibition of SFKs and the formation of N-cadherin cell-cell junctions. (+info)