Figure 3
A HIRA- and transcription- dependent H3.3 incorporation onto transplanted chromatin is required for transcriptional reprogramming following nuclear transfer. (A) ChIP analysis of H3.2 and H3.3 incorporation onto transplanted chromatin following nuclear transfer to oocytes under conditions where transcription or HIRA is inhibited. Recipient oocytes were preinjected with either no mRNA, HA-H3.2 mRNA, or HA-H3.3 mRNA. RA-ESC #5 nuclei were then injected in the GV with or without co-injection of either alpha amanitin (RNA Polymerase II inhibition) or anti-HIRA Antibody (HIRA inhibition). Twenty-four hours after transplantation H3.2 and H3.3 incorporation onto various genomic regions was measured by ChIP analysis. Error bars indicate s.e.m. (n = 3). (B) Confocal analysis of H3.3 accumulation onto transplanted chromatin following nuclear transfer to oocytes in conditions where transcription or HIRA is inhibited. Oocytes were preinjected with H3.3-GFP mRNA. H3.2-cherry expressing ESC #5 nuclei were then transplanted to the oocyte in the presence of either alpha amanitin (RNA Polymerase II inhibition), anti-HIRA Antibody (HIRA inhibition), or control Antibody. Forty-eight hours after transplantation, H3.3-GFP loading onto chromatin was analyzed by confocal imaging of isolated GVs. Graphs show fluorescence intensity of donor H3.2-cherry (red) and oocyte H3.3-GFP (green) across a section of the image (black arrow). (C) qRT-PCR analysis of transcription 24 h following nuclear transfer of RA-ESC #5 nuclei in the presence of either alpha amanitin (RNA Polymerase II inhibition), anti-HIRA Antibody (HIRA inhibition), or control Antibody. Error bars indicate s.e.m. (n = 3). (D) Western blot analysis of xenopus HIRA expression 48 h following injection of HIRA mRNA to the oocytes. (E) qRT-PCR analysis of transcription 24 h following the transfer of RA-ESC #5 nuclei in the presence or absence of anti-HIRA antibody in GVs of oocytes pre-injected with various doses of HIRA mRNA. Error bars indicate s.e.m. (n = 3). ★ indicate P value < 0.05, (T-TEST).