To gain further insight into the mechanisms by which histone citrullination may regulate gene activity, we also tested whether Cl amidine affected levels of di methylated histone H3K9, a modification screening libraries closely asso ciated with transcriptional repression. We found that levels of this modification were not significantly affected by Cl amidine treatment. To validate the specificity of the acetylated histone anti bodies, the embryos were also treated with the HDAC inhibitor Trichostatin A, and as expected, staining for acetylated histones was elevated following TSA treatment. Taken together, these results suggest that in hibition of PADI mediated histone citrullination sup presses histone acetylation.

Histone hyperacetylation promotes histone citrullination in early embryos To further explore the potential interplay between histone citrullination and acetylation, the colocalization of citrulli nation at H3R2 8 17 and acetylation at H3K9 was tested in 2 cell embryos. Results revealed that these two histone modifications localize, in part, to different regions of the nucleus, and they do appear to colocalize at specific foci in 2 cell embryos. To further test whether there was a potential crosstalk between his tone citrullination and acetylation, levels of H3R2 8 17 citrullination were examined in embryos treated with either TSA or Cl amidine. To perform this experiment, PN zygotes were recovered from B6D2F1/J females and cul tured in KSOM medium supplemented with either 100 nM of TSA or 250 uM of Cl amidine, respectively, for 68 hours.

Results showed that, as expected, levels of H3Cit2 18 17 were significantly reduced following Cl amidine treatment. Interestingly, however, we found that induction of histone hyperactylation via TSA treatment also resulted in significantly increased levels of citrullination at H3R2 8 17. This observation further suggests that there is a reinforcing relationship between acetylation and citrullination and further highlights the po tential interplay between these two modifications on chro matin in preimplantation mammalian embryos. Conclusions This report is the first to document the presence of citrullinated histones in mammalian oocytes and pre implantation 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 Brefeldin_A 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 po tentially 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 his tone H3K9 dimethyl modification.