Project description:Poly(ADP-ribose) polymerase 3 (PARP3) is a member of the PARP family of enzymes. It is structurally related to the well characterized type member PARP1 that orchestrates DNA strand break repair and cell death by the synthesis of poly(ADP-ribose). In contrast, the functions of PARP3 are undefined. We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. A ChIP-chip analysis of PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH reveals that PARP3 is preferentially associated with developmental genes and is significantly enriched around the transcriptional start site of several genes encoding neuronal and sensory placodes specifiers, such as SOX9, DLX3 and DLX4. Using zebrafish as a vertebrate animal model, we demonstrate that Parp3 plays a key role in ectodermal and neural crest specification. Morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud. Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border. ChIP were conducted as described (Rodrigue et al. 2006). PARP3 was immunoprecipitated from 3 x 10^7 SK-N-SH cells with a commercial anti-PARP3 (Enzo Life Sciences ALX-210-541). In control ChIP, PARP3 antibodies were replaced by rabbit IgG. Two independent PARP3 and control ChIP-chip experiments were achieved. The DNA was amplified using LM-PCR and labelled with Cy5 (PARP3) and Cy3 (IgG) before hybridization on Agilent Human promoter arrays (two 244k chip per ChIP for a total of 4 chips). Detailed protocols can be found at http://www.ircm.qc.ca/microsites/francoisrobert/en. Regions significantly enriched for PARP3 relative to IgG were identified as described (Lee et al. 2006).

Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other

Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA).

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Comparative RNAseq of Mouse Parp3+/+ and Parp3-/- Neural Stem Progenitor Cells (NPSC)

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