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JJ Jadasz FJ Rivera A Taubert M Kandasamy B Sandner N Weidner O Aktas HP Hartung L Aigner P Küry 《Development (Cambridge, England)》2012,139(18):3306-3315
Our recent studies revealed p57kip2 as an intrinsic regulator of late gliogenesis and demonstrated that in oligodendroglial precursor cells p57kip2 inhibition leads to accelerated maturation. Adult neural stem cells have been described as a source of glial progenitors; however, the underlying mechanisms of cell fate specification are still poorly understood. Here, we have investigated whether p57kip2 can influence early events of glial determination and differentiation. We found that Sox2/GFAP double-positive cells express p57kip2 in stem cell niches of the adult brain. Short-hairpin RNA-mediated suppression of p57kip2 in cultured adult neural stem cells was found to strongly reduce astroglial characteristics, while oligodendroglial precursor features were increased. Importantly, this anti-astrogenic effect of p57kip2 suppression dominated the bone morphogenetic protein-mediated promotion of astroglial differentiation. Moreover, we observed that in p57kip2 knockdown cells, the BMP antagonist chordin was induced. Finally, when p57kip2-suppressed stem cells were transplanted into the adult spinal cord, fewer GFAP-positive cells were generated and oligodendroglial markers were induced when compared with control cells, demonstrating an effect of in vivo relevance. 相似文献
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The type I receptor-like protein tyrosine kinase MuSK is essential for the neuromuscular junction formation. MuSK expression is tightly regulated during development, but the underlying mechanisms were unclear. Here we identified a novel mechanism by which MuSK expression may be regulated. A cyclic AMP response element (CRE)-like element in the 5'-flanking region of the MuSK gene binds to CREB1 (CRE-binding protein 1). Mutation of this element increases the MuSK promoter activity, suggesting a role for CREB1 in attenuation of MuSK expression. Interestingly, CREB mutants unable to bind to DNA also inhibit MuSK promoter activity, suggesting a CRE-independent inhibitory mechanism. In agreement, CREB1 could inhibit a mutant MuSK transgene reporter whose CRE site was mutated. We provide evidence that CREB interacts directly with MyoD, a myogenic factor essential for MuSK expression in muscle cells. Suppression of CREB expression by small interfering RNA increases MuSK promoter activity. These results demonstrate an important role for CREB1 in the regulation of MuSK expression. 相似文献
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Reynaud EG Leibovitch MP Tintignac LA Pelpel K Guillier M Leibovitch SA 《The Journal of biological chemistry》2000,275(25):18767-18776
Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoD(Ser200) in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57(Kip2), MyoD(Ala200), a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoD(Ala200) alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57(Kip2) increases the levels of MyoD(Ala200) in cotransfected cells. This result implies that p57(Kip2) may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57(Kip2) increased the half-life of MyoD(Ala200). This increased half-life of MyoD involves a physical interaction between MyoD and p57(Kip2) but not with p16(Ink4a), as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57(Kip2) from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH(2) domain of p57(Kip2) associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH(2) terminus of p57(Kip2) identified the intermediate alpha-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the alpha-helix domain of p57(Kip2), which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57(Kip2) family members positively act on myogenic differentiation. 相似文献
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Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin 总被引:1,自引:0,他引:1
The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure. 相似文献
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The interphase nucleus and nuclear envelope can acquire a myriad of shapes in normal or pathological cell states. There exist
a wide variety of indentations and invaginations, of protrusions and evaginations. It has been difficult to classify and name
all of these nuclear shapes and, consequently, a barrier to understanding the biochemical and biophysical causes. This review
focuses upon one type of nuclear envelope shape change, named “nuclear envelope-limited chromatin sheets” (ELCS), which appears
to involve exaggerated nuclear envelope growth, carrying with it one or more layers of ∼30 nm diameter heterochromatin. A
hypothesis on the formation of ELCS is proposed, relating higher order heterochromatin structure in an interphase nucleus,
nuclear envelope growth, and nuclear envelope-heterochromatin interactions. 相似文献
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Recent epigenomic studies have predicted thousands of potential enhancers in the human genome. However, there has not been systematic characterization of target promoters for these potential enhancers. Using H3K4me2 as a mark for active enhancers, we identified genome-wide EP interactions in human CD4+ T cells. Among the 6 520 long-distance chromatin interactions, we identify 2 067 enhancers that interact with 1 619 promoters and enhance their expression. These enhancers exist in accessible chromatin regions and are associated with various histone modifications and polymerase II binding. The promoters with interacting enhancers are expressed at higher levels than those without interacting enhancers, and their expression levels are positively correlated with the number of interacting enhancers. Interestingly, interacting promoters are co-expressed in a tissue-specific manner. We also find that chromosomes are organized into multiple levels of interacting domains. Our results define a global view of EP interactions and provide a data set to further understand mechanisms of enhancer targeting and long-range chromatin organization. The Gene Expression Omnibus accession number for the raw and analyzed chromatin interaction data is . GSE32677相似文献
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DNase I digestion reveals alternating asymmetrical protection of the nucleosome by the higher order chromatin structure 总被引:2,自引:0,他引:2
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Staynov DZ 《Nucleic acids research》2000,28(16):3092-3099
DNase I was used to probe the higher order chromatin structure in whole nuclei. The digestion profiles obtained were the result of single-stranded cuts and were independent of pH, type of divalent ion and chromatin repeat length. Furthermore, the protection from digestion of the DNA at the entry/exit points on the nucleosome was found to be caused not by the H1/H5 histone tails, but by the compact structure that these proteins support. In order to resolve symmetry ambiguities, DNase I digestion fragments over several nucleosome repeat lengths were analysed quantitatively and compared with computer simulations using combinations of the experimentally obtained rate constants (some of which were converted to 0 to simulate steric protection from DNase I digestion). A clear picture of precisely defined, alternating, asymmetrically protected nucleosomes emerged. The linker DNA is inside the fibre, while the nucleosomes are positioned above and below a helical path and/or with alternating orientation towards the dyad axis. The dinucleosomal modulation of the digestion patterns comes from alternate protection of cutting sites inside the nucleosome and not from alternating exposure to the enzyme of the linker DNA. 相似文献
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Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin 总被引:2,自引:0,他引:2
70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect. 相似文献
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Nikola Lukic Stefanie Lapetina Hanna Grobe Kolluru D. Srikanth Shams Twafra Jonathan Solomon Tal Sneh Michal Gendler Ronen Zaidel-Bar Hava Gil-Henn 《Molecular biology of the cell》2021,32(21)
Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2–/– mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2–/– MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein–protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2–/– MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand. 相似文献
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Roles of H1 domains in determining higher order chromatin structure and H1 location 总被引:29,自引:0,他引:29
J Allan T Mitchell N Harborne L Bohm C Crane-Robinson 《Journal of molecular biology》1986,187(4):591-601
Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones. 相似文献
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Expansion of chicken erythrocyte nuclei upon limited micrococcal nuclease digestion. Correlation with higher order chromatin structure 总被引:2,自引:0,他引:2
J E Hyde 《Experimental cell research》1982,140(1):63-70
A sensitive method for measuring nuclear volumes with a Coulter counter is described. It has been applied to the digestion of chicken erythrocyte nuclei by micrococcal nuclease and DNase I. Early in digestion, micrococcal nuclease induced a 20% increase in the effective spherical volume of the nuclei, followed by a gradual reduction. At the peak of nuclear swelling, about 17% of the chromatin was soluble after lysis and its average chain length was about 18 kilobase pairs (kb). DNase I digestion did not give rise to a corresponding expansion of the nuclei. Several preparation conditions, including the treatment of nuclei with 0.2% Triton X-100, led to a loss of the expansion effect upon subsequent micrococcal nuclease digestion. The results support the domain theory of higher order chromatin structure. In the context of this model, the observed maximum nuclear expansion correlates with an average of one nuclease scission per domain. 相似文献
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