Unknown,Transcriptomics,Genomics,Proteomics

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How basic nuclear processes are affected by selected HAT and HDAC inhibitors - Signalling pathways affected by HATs/HDACs inhibitor

ABSTRACT: Balance between histone acetylation and deacetylation is fundamental for proper gene function, thus knowledge of how to modulate these epigenetic processes is important for understanding of nuclear events, including replication, transcription, splicing and DNA repair. Here, we have studied an effect of HDAC inhibitor, SAHA, and we compared it with an effect of natural product, derivative of anacardic acid (MG149). Recently, it was discovered that MG149 has inhibitory potential for HATs' activity on histone H3 and H4 when tested on nuclear extracts.

ORGANISM(S): Homo sapiens

SUBMITTER: Hynek Strnad

PROVIDER: E-MTAB-1481 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

Project description:Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 Neural Precursor cells Embryonic stem (ES) cell lines provide, theoretically, unlimited access to any needed amount of any specific cell phenotype of an organism, due to their unique capacities at indefinite self-renewal and pluripotency (Smith 2001; Trounson 2006). These properties allow using the progeny of ES cell lines to model human pathologies (Martinat, Shendelman et al. 2004; Lerou and Daley 2005; Ben-Nun and Benvenisty 2006). In particular, human ES cell lines derived from embryos characterized as gene-carriers following pre-implantation genetic diagnosis (PGD) for any one of major monogenic diseases (Pickering, Minger et al. 2005; Mateizel, De Temmerman et al. 2006) may be considered as perfect cellular replicas of those diseases, as they exhibit the exact genotypes associated to them. Here, we confirm this hypothesis by demonstrating that the cell progeny of an ES cell line derived from an embryo with myotonic dystrophy type 1 (DM1) displayed the morphological stigma associated to the expression of the mutant gene âso-called intranuclear foci- as well as abnormal alternate splicing of the insulin receptor, a characteristic feature of DM1. Further differential transcriptomic analysis of the DM1 gene-carrying cells with phenotypically similar populations from native ES cell lines revealed abnormal expression of 89 genes, among which 48 were down-regulated and 39 over-expressed. This study demonstrates that DM1, though a disease with relatively late clinical onset, is associated with expression of genetic defects early on during development. It underlines the value of PGD-derived ES cell lines as a tool to decipher molecular mechanisms of genetic diseases. Ben-Nun, I. F. and N. Benvenisty (2006). Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252(1-2): 154-9. Lerou, P. H. and G. Q. Daley (2005). Therapeutic potential of embryonic stem cells. Blood Rev 19(6): 321-31. Martinat, C., S. Shendelman, et al. (2004). Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism. PLoS Biol 2(11): e327. Mateizel, I., N. De Temmerman, et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21(2): 503-11. Pickering, S. J., S. L. Minger, et al. (2005). Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod Biomed Online 10(3): 390-7. Smith, A. G. (2001). Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17: 435-62. Trounson, A. (2006). The production and directed differentiation of human embryonic stem cells. Endocr Rev 27(2): 208-19. Keywords: disease state analysis Two controls hES-derived NPC (VUB01 and SA01) and one mutant hES-derived NPC (VUB03_DM1), with three biological replicats for each

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How basic nuclear processes are affected by selected HAT and HDAC inhibitors - Signalling pathways affected by HATs/HDACs inhibitor

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