Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline1. This modification leads to the loss of a positive charge and reduction in hydrogen bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs)2 and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis2,3. Nonetheless, the physiological functions of citrullination remain ill-defined, though citrullination of core histones has been linked to transcriptional regulation and the DNA damage response4–8. PADI4 (or PAD4/PADV), the only PADI with a nuclear localization signal9, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection10. Here we show that the expression and enzymatic activity of PADI4 are also induced under conditions of ground state pluripotency and during reprogramming. PADI4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin11, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

SummaryWe constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three-to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.

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 oocytespecific 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. chromatin | nuclear transfer | Xenopus T he transfer of somatic cell nuclei to eggs is an experimental means of reversing the process of cell differentiation in which cells become progressively restricted in the developmental pathways open to them (1-3). By comparison with other procedures (4, 5), nuclear transfer (NT) is relatively efficient (6), and it makes use of natural components of eggs without any accompanying change to the genome. A variant of this technique is NT to Xenopus oocytes (7). Although no new cell types are generated in this type of NT, reactivation of pluripotency genes takes place within a day after NT and in the absence of cell division. The oocyte (M1 prophase I) is the immediate progenitor of an egg (M2 metaphase) and is believed to reprogram transcription in the same way that an egg reprograms the sperm nucleus after fertilization. Therefore the direct and efficient transcriptional reprogramming activity of the Xenopus oocyte makes it a favorable cell in which to analyze an important part of the mechanism of nuclear reprogramming.Our aim is to understand the mechanism of reprogramming by NT in eggs and oocytes. The substrate for this reprogramming activity is the chromatin of transplanted nuclei. The chromatin of eukaryotes contains DNA wrapped around the four core histones arranged as a nucleosome. The linker DNA joining two nucleosomes is also bound by chromatin proteins such as linker histones, high mobility group proteins (8, 9), and poly (ADPribose) polymerase 1 (10). Several linker histone variants are present in somatic cells, and the ratio of the various forms varies from one cell type to another (11). Linker histones initially were thought to have a general function in repressing gene activity. Recent work has demonstrated that linker...

Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.The remarkable stability of cell differentiation under normal conditions can be reversed experimentally by nuclear transfer, cell fusion and induced pluripotent stem (iPS) cell technology [1][2][3][4][5] . This provides an opportunity to generate pluripotent embryonic cells from adult cells of the same individual and hence opens the possibilit y of cell replacement without the need for immunosuppression.iPS cell technology makes use of the overexpression of transcription factors (FIG. 1a) and has been extensively reviewed, so it is not discussed in detail [6][7][8][9] . To generate entirely unrelated cell types by iPS cell technology, treated cells must be grown for multiple cell divisions over a long period of time (up to 3 weeks). By contrast, nuclear transfer and cell fusion do not involve the overexpression of new genes, and instead make use of natura components present in eggs and some early embryos to initiate new transcription.There are two kinds of nuclear transfer experiments: egg-NT involves the transfer of a single somatic nucleus to an unfertilize d enucleated egg (in both mammals and amphibians) 1 (FIG. 1b); and ooc-NT involves the transplantation of multiple somatic cell nuclei into the germinal vesicle (the nucleus) of a growing meiotic prophase amphibian oocyte (an immature egg) 10 (FIG. 1c). Note that the terms egg and oocyte refer to different developmental stages in amphibians and mammals: amphibian eggs are in metaphase II of meiosis, which is equivalent to mouse metaphase II stage oocytes, whereas their immediate precursors, oocytes, are blocked in meiotic prophase I, which is equivalent to mouse germinal vesicle stage oocytes.© 2011 Macmillan Publishers Limited. All rights reserved Correspondence to J.B.G.j.gurdon@gurdon.cam.ac.uk. Competing interests statementThe authors declare no competing financial interests. There are important differences between the two types of nuclear transfer experimen t. Extensive cell division takes place in egg-NT experiments, and functional new cell types appear as the nuclear transplant embryo develops. By contrast, in ooc-NT experiments, no new cell types are formed, and neither the oocyte nor the introduced nuclei divide, but there is a direct transition from nuclei of differentiated cells to reprogrammed nuclei that transcribe pluripotency genes. Analysis of the mechanism of reprogramming (which involves transcription of pluripotency and other genes) in egg-NT expe...