[show abstract][hide abstract] ABSTRACT: Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)
[show abstract][hide abstract] ABSTRACT: Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.
Molecular and Cellular Biology 06/2000; 20(9):3316-29. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.
Annals of the New York Academy of Sciences 10/1999; 883:234-46. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Leukemia inhibitory factor (LIF) regulates the mature hypothalamic-pituitary-adrenal axis in vivo. In vitro, LIF determines corticotroph cell proliferation and induces POMC transcription. To explore LIF action on pituitary development, transgenic mice expressing LIF driven by the pituitary glycoprotein hormone alpha-subunit (alphaGSU) promoter were generated. Transgenic mice exhibited dwarfism with low IGF-I (29 +/- 9 ng/ml vs. wild type (WT) 137 +/- 16 ng/ml; P < 0.001), hypogonadism with low FSH (0.04 +/- 0.023 ng/ml vs. WT 0.63 +/- 0.18 ng/ml; P < 0.001), and Cushingoid features of thin skin and truncal obesity with elevated cortisol levels (86 +/- 22 ng/ml vs. WT 50 +/- 14 ng/ml; P = 0.002). Their pituitary glands showed corticotroph hyperplasia, striking somatotroph and gonadotroph hypoplasia, and multiple Rathke-like cysts lined by ciliated cells. LIF, overexpressed in Rathke's pouch at embryonal day 10, diverts the differentiation stream of hormone-secreting cells toward the corticotroph lineage and ciliated nasopharyngeal-like epithelium. Thus, inappropriate expression of LIF, a neuro-immune interfacing cytokine, plays a key role in the terminal differentiation events of pituitary development and mature pituitary function.
[show abstract][hide abstract] ABSTRACT: Leukemia inhibitory factor (LIF) and LIF receptors are expressed in adenohypophyseal cells and LIF regulates pituitary hormone transcription and cell replication in vitro. Therefore, transgenic mice expressing pituitary-directed LIF driven by the rat growth hormone (GH) promoter were generated to evaluate the impact of LIF on pituitary development. Three founders were established with diminished linear growth and body weight (57-65% of wild type [WT]), and intense anterior pituitary LIF immunoreactivity. Cystic cavities observed in pituitary anterior lobes were lined by cuboidal, ciliated epithelial cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones. Transgenic pituitaries showed decreased GH (40%) and prolactin (PRL) (26%) cells, and decreased GH and PRL mRNAs by in situ hybridization. ACTH cells increased 2.2-fold, whereas gonadotrophs and thyrotrophs were unchanged. Serum GH was undetectable (< 0.78 ng/ml), PRL levels were one third of WT (P < 0.05), IGF-I levels were 30% of WT (P < 0. 001), and T4 was normal. 10 human pituitary Rathke's cysts studied all showed conclusive LIF immunoreactivity in cyst-lining cells. Thus, intrapituitary murine LIF overexpression causes cystic invaginations from the anterior wall of Rathke's cleft, suggesting failed differentiation of Rathke's epithelium to hormone-secreting cells. Arrested murine pituitary maturation with formation of pituitary Rathke's cleft cysts, GH deficiency, and short stature provide a model to study human Rathke's cyst pathogenesis.
Journal of Clinical Investigation 06/1997; 99(10):2462-9. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.
Proceedings of the National Academy of Sciences 06/1995; 92(10):4447-51. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteolipid protein (PLP) is an integral membrane protein of CNS myelin. Mutations of the X chromosome-linked PLP gene cause glial cell death and myelin deficiency in jimpy mice and other neurological mutants. As part of an attempt to rescue these mutants by transgenic complementation, we generated normal mouse lines expressing autosomal copies of the entire wild-type PLP gene. Surprisingly, increase of the PLP gene dosage in nonmutant mice with only 2-fold transcriptional overexpression results in a novel phenotype characterized by severe hypomyelination and astrocytosis, seizures, and premature death. This demonstrates that precise control of the PLP gene is a critical determinant of terminal oligodendrocyte differentiation. Dysmyelination of PLP transgenic mice provides experimental evidence that Pelizaeus-Merzbacher disease, previously associated with a partial duplication of the human X chromosome, can be caused by doubling of the PLP gene dosage.
[show abstract][hide abstract] ABSTRACT: Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.
The Journal of Cell Biology 11/1993; 123(2):443-54. · 10.82 Impact Factor