[show abstract][hide abstract] ABSTRACT: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated.
In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17.
Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.
[show abstract][hide abstract] ABSTRACT: Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.
[show abstract][hide abstract] ABSTRACT: Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton) insoluble structures termed the oocyte cytoplasmic lattices (CPLs). Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function.
Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater(tm/tm) oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater(+/+) and Mater(tm/tm) germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/tm) oocytes compared to Mater(+/+) oocytes.
Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.
PLoS ONE 01/2010; 5(9):e12587. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna(-/-)), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna(-/-)tg(K164R)) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna(-/-)tg(K164R) mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna(-/-)tg(K164R) mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-eta (Poleta), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna(-/-)tg(K164R) mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).
Proceedings of the National Academy of Sciences 11/2008; 105(42):16248-53. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.
Biology of Reproduction 04/2008; 78(3):462-71. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.
Proceedings of the National Academy of Sciences 07/2004; 101(23):8652-7. · 9.74 Impact Factor