Potential role for PADI-mediated histone citrullination in preimplantation development

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Temporal and spatial distribution of histone H3 and H4 citrullination in oocytes and preimplantation embryos. A. Oocytes and embryos were isolated from CD1 female mice, fixed, permeabilized, and immunolabeled with antibodies against H4Cit3 (red). B. Same as (A) except anti-H3Cit2 + 8 + 17 antibodies (red) were used. Spindle apparatus and microtubule based mid-bodies are indicated by arrows and arrowheads, respectively. C. Same as (A) except anti-H3Cit26 antibodies (red) were used. Nucleoli are denoted by arrows. All samples were counterstained with 4′,6-diamidino-2-phyenylindole (DAPI) to visualize DNA (Blue). Cells were imaged by laser scanning confocal microscopy. DIC, differential interference contrast. PN, pronuclear. Blast., blastocyst.

Figure 2

Cl-amidine blocks embryonic developmentin vitroat the 2 to 4 cell stage. A. Immunofluorescent confocal images showing that Cl-amidine reduces citrullination at H4R3 in 4-cell embryos. PN stage zygotes were cultured for ~42 hours or ~68 hours in KSOM medium supplemented without or with 250 μM of Cl-amidine. Four-cell embryos were fixed and immunostained with anti-H4Cit3 antibodies (red). Phalloidin and DAPI were employed to detect F-actin (red) and DNA (blue). Arrows highlight H4Cit3 staining on chromatin. DIC, differential interference contrast. B. Same as (A) except with anti-H3Cit2 + 8 + 17 antibodies. C. Same as (A) except with anti-H3Cit26 antibodies. D. Structure of Cl-amidine. The red square highlights the chlorine group that is replaced with hydrogen to generate H-amidine. MW., Molecular weight. E. Structure of H-amidine. F. Rate of development to the morula stage following Cl-amidine treatment. Pronuclear stage zygotes were cultured for ~68 hours in KSOM medium supplemented with 250 μM of Cl-amidine or H-amidine and observed by light microscopy. H-AM, H-amidine.

Figure 3

Effect of Cl-amidine and TSA treatment on histone H3 and H4 acetylation and methylation. A. PN zygotes were cultured for ~68 hours in KSOM medium supplemented with either 250 μM of Cl-amidine or 100 nM of TSA. Two or four-cell embryos were fixed and immunostained with antibodies to hyperacetylated H4 (red) and acetyl H3K9 (green). DAPI was utilized to detect DNA (blue). DIC, differential interference contrast. B. Histograms documenting the fluorescent intensity of the hyperacetylated H4 and acetyl H3K9 signals shown in A. C. Same as (A) except antibodies against acetyl H3K18 (red) and di-methyl H3K9 (green) were used. D. Histograms documenting the fluorescent intensity shown in B experiments. E. Same as (A) except antibodies against acetyl H4K5 (red) were used. F. Histogram documenting the fluorescent intensity in experiment shown in E. Data are presented as mean + SEM. *P < 0.05, **P < 0.005, ***P < 0.001 (two-tailed paired Student's t-test).

Figure 4

Potential crosstalk between histone citrullination and acetylation in early embryos. A. Colocalization of citrullinated H3R2 + 8 + 17 and acetylated H3K9. Two-cell embryos were immuno-stained with antibodies to H3Cit2 + 8 + 17 (red) and H3acK9 (green). The colocalization is highlighted in yellow in the merged panel. B. PN stage zygotes were co-cultured for ~68 hours in KSOM medium with 250 μM of Cl-amidine or 100 nM of TSA. Two or four-cell embryos were fixed and immuno-stained with antibodies to H3Cit 2 + 8 + 17 (red). DAPI was utilized to detect DNA (blue). DIC, differential interference contrast. Data are presented as mean + SEM. *P < 0.05, (two-tailed paired Student's t-test).