Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation.
When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.
Jullien
Jerome
J
The Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, United Kingdom.
Astrand
Carolina
C
Halley-Stott
Richard P
RP
Garrett
Nigel
N
Gurdon
John B
JB
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D055748:SOXB1 Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D029867:Xenopus Proteins; C527781:sox2 protein, Xenopus; D014212:Tretinoin