Citrullination regulates pluripotency and histone H1 binding to chromatin. Christophorou MA 2014 et al.
Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. 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) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. 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 chromatin, 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.
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NCBI Summary:
This gene is a member of a gene family which encodes enzymes responsible for the conversion of arginine residues to citrulline residues. This gene may play a role in granulocyte and macrophage development leading to inflammation and immune response. [provided by RefSeq, Jul 2008]
General function
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Cellular localization
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Ovarian function
Early embryo development
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SSR 2010 Peptidylarginine deiminases are a family of calcium dependent, highly substrate
specific enzymes that convert peptide bound arginine residues into non conventional
amino acid citrulline in a post translational modification known as citrullination.
Among the known 5 isomers, peptidylarginine deiminase 4 (PAD4) having nuclear
localizing sequence, is localized in the nucleus and is known to target histone and
nucleophosmin. It has been shown that modifications mediated by PAD4 are
involved in regulating chromatin structure and gene expression in a variety of cell
types. Recent studies have confirmed regulation of p53 target gene expression by
peptidylarginine deiminase 4. Here we study the localization and expression of PAD4
in pig sus scrofa oocytes and electrically activated preimplantation embryos and
analyze its role in the preimplantation development. 4-6 mm sized follicles were
extracted from the ovaries that were collected from the local slaughter house and
cultured for maturation in vitro. Oocytes were collected at different stages of
maturation for analysis. Fully matured metaphase oocytes with extruded 1st polar
body collected at 42-46 hrs post maturation culture were electrically stimulated. Thus
activated embryos were collected at different developmental stages for the
investigation. Indirect immunofluorescence assays and western blot analyses, showed
that PAD4 is localized in the cytosol in all the stages of oocytes and embryos.
However, in germinal vesicle oocyte, it was also found to have translocated into the
nucleus before germinal vesicle break down and then onwards it was detected to be
colocalized with the metaphase spindle. The association of PAD4 with chromatin was
observed until the completion of 2nd meiosis. PAD4 substrate specificity was
dissected using modified citrulline detection kit and it was discovered that histone
was the target of peptidylarginine deiminase 4 in oocytes and embryos. It was noticed
that the amount of histone citrullination was different in different stages of
development. There was a dip in the citrullination signal at 2 cell stage which
increased from 4 cell stage onwards, to be highest at the blastocyst stage. When
PAD4 expression was knocked down via RNA interference, it was observed that
there was delay in the development which lead to retarded growth. The delay was
noticed at each developmental stage. There was significant decrease in the blastocyst
cell number. When PAD4 expression and localization was also checked in mouse
oocytes and parthenogenetic embryos, metaphase oocytes showed similar PAD4
localization pattern and the PAD4 expression was seen in all the stages of embryos
too. To check whether oocyte associated somatic cells such as cumulus cells also
contain PAD4 and are involved in the oocyte development, cumulus cells collected
during denudation of metaphase oocytes were examined for the presence of PAD4 as
well as citrullinated proteins in the nucleus by both indirect immunofluorescence and
western blotting analyses. It was revealed that PAD4 was only cytoplasmic and
histone 3 was the target. Thus our study confirms the presence of PAD4 in the pig
oocytes, embryos and cumulus cells; PAD4 nuclear translocation in the later stages of
oocyte development and during fertilization; and leads to the conclusion that PAD4 is
involved in the development of pig preimplantation embryos.
cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI Zhang J, et al .
Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.
Expression regulated by
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Ovarian localization
Oocyte
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Localization and expression of peptidylarginine deiminase 4 (PAD4) in mammalian oocytes and preimplantation embryos. Brahmajosyula M et al. SummaryPost-translational modifications generally involve the addition or removal of various functional groups to or from the protein residues. However, citrullination, which is catalyzed by the peptidylarginine deiminases (PADs), involves conversion of one kind of amino acid residue into another. One of five isoforms, PAD4 is a nuclear enzyme known to play a role in development, differentiation and apoptosis through gene regulation. To investigate the possible role of PAD4 in mammalian preimplantation embryonic development, we first studied localization and expression of PAD4 and citrullinated proteins in pig and mouse oocytes, and parthenogenetic or in vitro fertilized (IVF) embryos. Immunofluorescence study revealed that PAD4 primarily localizes in the cytoplasm in pig oocytes and parthenogenetic embryos. However, the nuclear translocation of PAD4 was observed in late germinal vesicle (GV) stage oocytes prior to GV breakdown and was localized around the metaphase (M)I and II spindle. Nucleus localized PAD4 was noticed partially again in blastocysts. In mouse IVF embryos, nuclear translocation started from the 2-cell stage and gradually increased up to blastocyst. Western blot studies confirmed that PAD4 was expressed in oocytes, and parthenogenetic embryos of pig. Citrullinated proteins were detected in granular form on the chromatin in GV, MI and MII oocytes and nuclei in all the stages of the embryos studied. It was found that the target of citrullination was histone protein (H3), not B23. Therefore the presence of PAD4 and citrullinated histone H3 in oocytes and embryos suggested a possible role for PAD4 in preimplantation embryonic development.