Figure 2From: HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytesKinetics of transcription and H3.3 incorporation following nuclear transplantation to Xenopus oocytes. (A) Schematic representation of the Oct4 transgene composed of a 20 copy array in the embryonic stem cells used in this study (ESC #5). DE, Distal enhancer; PE, Proximal enhancer; PP, Proximal promoter. (B) qRT-PCR analysis of transcription during a 48-h period following nuclear transplantation of 4 day-retinoic acid differentiated ESC #5 nuclei. Error bars indicate s.e.m. (n = 3). (C) Analysis of rDNA and major satellite transcription rates. Oocytes were transplanted with RA-ESCs and injected with BrUTP at various times after nuclear transfer (at 0, 12, 24, or 36 h). Oocytes were then collected 12 h after the time of BrUTP injection. In that way, the different samples collected will contain transcripts produced by the transplanted nuclei and labeled by BrUTP during a 12-h period (T12 to 24 h, T24 to 36 h, T36 to 48 h). After mRNA extraction BrUTP labeled transcripts are purified by immunoprecipitation with an anti-BrdUTP antibody and analyzed by qRT-PCR. Error bars indicate s.e.m. (n = 3). (D) ChIP analysis of histone incorporation 24 h after NT in oocytes preinjected with mRNA for HA-H3.3 and HA-H3.2, performed over the Oct4 reporter promoter, major satellite and ribosomal DNA genes, respectively. The total amount of incorporated H3.3 is set to 1. Error bars indicate standard deviation (n = 3). (E) Analysis of histone incorporation kinetics over Oct4 regulatory regions after NT. Four-day retinoic acid treated ESC #5 were transplanted in oocytes preinjected with mRNA for HA-H3.3 and HA-H3.2, collected over a 48-h time course and analyzed by ChIP with an HA antibody.Back to article page