Potential role for PADI-mediated histone citrullination in preimplantation development
© Kan et al.;licensee BioMed Central Ltd. 2012
Received: 6 December 2011
Accepted: 23 May 2012
Published: 19 June 2012
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.
The fundamental repeating unit of chromatin is the nucleosome that contains two superhelical turns of DNA wrapped around an octamer of two copies each of the core histones H2A, H2B, H3 and H4 [1, 2]. Resolution of the nucleosome structure revealed that the N-terminal histone tails protrude from the nucleosomal core in an unstructured manner  and contain an ever-growing number of post-translational modifications such as acetylation, methylation, phosphorylation, and more recently, citrullination [4, 5]. Importantly, these modifications, or “marks”, play critical roles in many cellular functions, including DNA replication, condensation, and repair, as well as gene regulation . Of these modifications, histone acetylation is perhaps most strongly associated with gene regulation. Increasing levels of histone acetylation are correlated with a transcriptionally permissive state whereas deacetylated histone are closely associated with transcriptional repression . Histone acetylation is also implicated in the activation of embryonic gene expression in preimplantation embryos . For example, previous reports investigating late two-cell embryos have found that inducing histone hyperacetylation with HDAC inhibitors stimulates global transcription  and depletion of HDAC1 by RNAi results in elevated levels of specific gene targets . Previous studies in somatic cells have demonstrated that specific histone modifications can directly affect the levels of other marks and this interplay leads to a complex mechanism of gene regulation, frequently referred to as the “histone code” [10, 11]. While fewer of these types of studies have been carried out in early embryos, several reports have found that cross-talk exist between histone acetylation and histone methylation in normal and cloned embryos .
PADI enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. For example, we have found that PADI4-mediated citrullination of histone H4 arginine 3 at the TFF1promoter in MCF7 cells appears to regulate the expression of this canonical estrogen receptor target . Others have shown that PADI4-mediated histone citrullination plays a role in regulating other target genes such as TRP53 and OKL38[14, 15]. In addition to PADI4, we recently found that PADI2 localizes to the nucleus of mammary epithelial cells and appears to target histone H3 for citrullination , thus suggesting that multiple PADIs regulate chromatin-based activities.
We have previously documented that oocyte -and-embryo abundant PADI6 is required for female fertility, with PADI6-null embryos arresting at the two-cell stage of development . Given the growing body of literature linking PADI enzymes to histone citrullination and the abundance of PADI6 in oocytes and early embryos, we first tested whether histones were citrullinated in oocytes and preimplantation embryos. Next, we treated embryos with the PADI-specific inhibitor, Cl-amidine, to confirm that the observed citrulline marks were generated by PADI activity and also to test whether inhibition of PADI activity may affect preimplantation development in vitro. Lastly, to gain insight into potential mechanisms by which histone citrullination may regulate gene activity, we tested whether inhibition of PADI activity in early embryos affects histone acetylation and whether induction of histone hypoacetylation affected levels of histone citrullination. Findings from this study are discussed below.
Results and discussion
Citrullination of histone H3 and H4 tails in oocytes and in preimplantation embryos appears to be robust and dynamic
The staining pattern observed with the anti-H3Cit26 was perhaps the most interesting of all. Results found that both oocytes and early embryos showed a strong punctate cytoplasmic signal that appeared to coalesce into larger aggregates as the embryos developed (Figure 1C) and these foci appeared by light microscopy to be lipid droplets. To test this hypothesis we stained mutant MATER GV oocytes (which have elevated levels of lipid droplets ) with Nile Red or with the H3Cit26 antibody. Results show (Additional file 1) that the H3Cit26-containing cytoplasmic foci clearly appear to be lipid droplets. Interestingly, a recent study found that lipid droplets in Drosophila embryos are maternally-derived and that these structures contain ~50% of all embryonic histones. This finding suggests that the lipid droplets function to sequester maternal histones in the early embryo until they are needed for chromatin-based activities . An intriguing possibility is that the H3Cit26 modification marks histones for lipid droplet storage and/or possibly shuttling histones between lipid droplets and the nucleus. H3Cit26 staining within the nucleus was also interesting; whereas little to no signal was seen in GV stage oocytes, strong staining was observed on the outer margins of both male and female pronuclei and around the nucleoli of two-cell embryos (Figure 1C; arrows in panels of 2-PN and 2-cell). By the four-cell stage of development no nuclear staining was observed. Given that embryonic genome activation is known to initiate at the late pronuclear/early two-cell stage, this observation raises the possibility that this particular citrulline modification may play a role in activation of the embryonic genome. Taken together, our data raise the possibility that the different histone modification sites may play different roles in preimplantation development.
Given that each of these anti-citrullinated histone antibodies showed both cytoplasmic and nuclear localization patterns, we next confirmed the specificity of our antibodies by testing whether pre-absorption of the antibodies with their cognate peptide affected indirect immunofluorescence signal intensity levels (Additional file 2). Results showed that peptide preabsorption suppressed the fluorescence intensity for each of the three antibodies (Additional files 2A, 2B, and 2C, respectively). These results suggest that the localization patterns observed for the H4Cit3, H3Cit2 + 8 + 17, and H3Cit26 are specific.
Cl-amidine blocks mouse embryonic development beyond the two to four cell stage in vitro
The above observations suggested that PADI-mediated histone citrullination may play an important, previously unknown, role in early development. Given that PADI6 is essential for early cleavage divisions, we next tested whether levels of these modifications were reduced in PADI6-null mouse oocytes/early embryos. We found that loss of PADI6 did not appear to affect histone citrullination levels (see Additional file 3A, 3B, and 3C). Given PADI4’s previously documented roles in histone citrullination and gene regulation [13, 14], we then tested citrullinated histone levels in PADI4-null oocytes. Again, we did not observe any appreciable loss in levels of citrullinated histone in this mutant line (see Additional file 4A and 4B). Together, these observations suggest, neither PADI4 nor PADI6 catalyze these specific citrulline modifications on histones in oocytes or early embryos.
The effect of Cl-amidine on early embryonic development
Stage of embryos (%)
Treatment of embryos with C-amidine suppresses histone H3 and H4 acetylation while having no apparent effect on the repressive H3K9 Di-methyl modification
Histone hyperacetylation promotes histone citrullination in early embryos
This report is the first to document the presence of citrullinated histones in mammalian oocytes and preimplantation embryos. The use of three site-specific citrullinated histone antibodies found that histone citrullination is likely playing several unique, yet to be defined roles on chromatin templated events. We found that the PADI inhibitor, Cl-amidine, potently blocks embryonic development beyond the 4-cell stage, thus further highlighting the important role of PADIs in early development. This observation also raises the possibility that PADI inhibitors could potentially be utilized as novel contraceptives. Our study also showed that Cl-amidine specifically suppressed histone acetylation on the H3 and H4 tails while not affecting levels of the transcriptionally repressive histone H3K9 dimethyl modification. Further, we found that induction of histone hyperacetylation leads to enhanced histone citrullination. Mechanistically, as with numerous other histone modifications [10, 11], these observations raise the distinct possibility that the citrulline modification on histones may function as a “platform” for binding by histone acetyltransferases (HATs), thus facilitating transcriptional activation by enhancing levels of histone acetylation. More detailed studies are now required to test this hypothesis. We predict that outcomes from the current study will likely lead to new and important insight into epigenetic regulation of the oocyte to embryo transition.
The generation of mouse mutants and genotyping strategies for the Padi6 and Mater null strain has been described previously [17, 25]. To generate Padi4-null mice, the entire genomic sequence of Padi4 was replaced in frame with the coding sequence of LacZ and a Lox-flanked neomycin gene driven by PGK-EM7 promoter. B6D2F1/J and CD-1 mice were purchased from the Jackson Laboratory and Charles River Laboratories, respectively. All mice were housed in the Cornell University Animal Facility (Ithaca, NY) and procedures using these mice were reviewed and approved by the Cornell University Institutional Animal Care and Use Committee. Studies were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Oocyte and embryo collection
All experiments were performed using B6D2F1/J and CD-1 female mice (age 4-8 weeks) primed with gonadotrophins (Sigma) to obtain fully-grown GV oocytes, ovulated oocytes, and embryos (following mating with CD1 males). All oocytes and embryos were collected in M2 medium (Sigma) unless otherwise stated. Culture medium was supplemented with 25 mM of milrinone (Sigma) to inhibit GVBD. Embryos at different developmental stages were collected and processed at different times.
Immunofluorescence and laser scanning confocal microscopy
Indirect immunofluorescence labeling confocal microscopy was undertaken as described previously . Rabbit-anti-H4Cit3 (1:50, Abcam), rabbit-anti-H3Cit2 + 8 + 17 (1:50, Abcam), rabbit-anti-H3Cit26 (1:50, Abcam), rabbit-anti-hyper acetyl H4 (1:50, Millipore), mouse-anti-acetyl H3K9 (1:20, Abcam), rabbit-anti-acetyl H4K5 (1:50, Abcam), rabbit-anti-acetyl H3K18 (1:50, Abcam), and mouse-anti-dimethyl H3K9 (1:20, Abcam) antibodies were used for this study. Images were obtained on LSM 510 laser scanning confocal microscopy (Carl Zeiss, Germany) equipped with Zen 2007 software for image processing. In the multicolor labeling experiments, the confocal configuration was set up to avoid the bleed-through of fluorescence dyes. To test for bleed-through, oocytes were stained with each dye separately and images were taken with multiple channels. Each signal was found to be well resolved from other signals. Images for each developmental series were collected under similar conditions using the following settings: ex = 555 nm, em = 565 nm, laser power 12%, frame size 1024 × 1024, scanning speed 7, averaging number 4, detector gain around 700, digital offset around -15, and Zen 2009 software. To directly compare changes in signal intensity for embryos from different treatment groups, confocal images were acquired under an identical condition for those samples.
Embryo culture and drug treatment
The KSOM medium for embryo culture has been described elsewhere .B6D2F1/J females were primed with 10 IU PMSG (Sigma) followed by 10 IU hCG (Sigma) injection, then housed with CD1 males. PN zygotes were collected in M2 medium at ~26 hours post hCG and cultured in KSOM for ~ 42 hours or 68 hours supplemented with 250 μM of Cl-amidine, 250 μM of H-amidine, or with 100 nM of TSA (Sigma). Embryos cultured at 37 °C in an atmosphere of 5% CO2, 5% O2 and 90% N2 were fixed and immunostained with antibodies at different time points for analyses. Digital images were recorded on the confocal microscopy.
Citrulline antibody absorption assay by antigen peptide
All antibodies and antigen peptides were purchased from Abcam with the exception of the H4Cit3 peptide which was a kind gift from David Allis at Rockefeller University. The ratios of antibody and peptide for H4Cit3, H3Cit2 + 8 + 17, and H3Cit26 were 1mol:20 mols, 1μl:6 μls, and 1mol:40mols, respectively. The antibody and peptide were added to the antibody dilution buffer (1% normal goat serum, 0.5% BSA in PBS) and incubated on a rotator at room temperature for 2 hours for the H4Cit3 and H3Cit2 + 8 + 17 mixtures or for 1 hour (4°C) for the H3Cit26 mixture.
Deionized water replaced the histone modification peptides as a control. GV-oocytes or 2-cell embryos were collected from CD1 females at ~46 hours post PMSG and ~ 46 hours post hCG and processed for immunofluorescence and laser scanning confocal microscopy as described above.
Nile red staining of mouse oocytes
Nile red powder (Sigma) was dissolved in DMSO to give a stock solution of 1mg/ml and stored at -20°C. GV-oocytes were collected in M2 media supplemented with IBMX from Mater mutant females. After three quick washes in PBS/PVA, oocytes were transferred into 4% paraformaldehyde/PBS and incubated for 30 min at room temperature. Oocytes were briefly washed three times in PBS/PVA again and transferred into nile red working solution (1μg/ml) for 30 min following the fixation. After the nile red staining, oocytes were washed three times and carefully added to the drop of Slowfade Gold antifade reagent (Molecular probe) on slides, and then a cover slide was placed on top of the drop. Nile red fluorescence was captured by Laser Scanning Confocal microscopy (Carl Zeiss).
Analysis of embryo viability
Pronuclear stage zygotes were retrieved from B6D2F1/J females at ~26 hours post hCG and cultured for ~68 hours in KSOM medium supplemented with 250 μM of Cl-amidine or H-AM. Embryos from these two groups were then incubated with 20 μg/ml of PI (Sigma) in KSOM for 5 min at 37 °C in an atmosphere of 5% CO2, washed 3 times, and images were recorded using Zeiss epifluorescence microscopy (Carl Zeiss, Germany). A sub set of Cl-amidine treated embryos were permeablized with 0.1% Triton for 20 min prior to PI staining to serve as positive control.
JC-1 staining of cultured embryos was performed according to the Invitrogen protocol. Briefly, embryos were stained with 10 μg/ml of JC-1(Invitrogen) in KSOM for 10 min at 37 °C in an atmosphere of 5% CO2, washed 3 times with KSOM, and images were captured by Zeiss epifluorescence microscopy using fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) filters.
All samples used for quantification included at least four nuclei. Data were compiled and analyzed using Microsoft Excel 2007. Comparisons were made for the intensity of histone modifications in embryos cultured in KOSM media supplemented with or without Cl-amidine using unpaired t-test. The threshold of statistical significance was set at P < 0.05.
This research was supported by NICHD grants RO1 38353 and RO3 522241 (S.A.C.) and by NIGMS grant GM079357 (P.R.T).
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