Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone metyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 causes diverse modifications to the chromatin during early development, suggesting an intense epigenetic crosstalk during nuclear reprogramming.

Cloning by somatic cell nuclear transfer (SCNT) is an inefficient technique largely because of 3 6 incomplete nuclear reprogramming. The persistent epigenetic memory from the somatic cell 3 7 nucleus is considered one of the main barriers for an efficient reprogramming process 1 . In most 3 8 cases, the recipient oocyte used in SCNT fails to erase residual epigenetic marks from somatic 3 9 nucleus, which are retained in the embryo and lead to abnormal gene expression 2 . For instance, gene expression during the period of embryonic genome activation (EGA) 3,4 . According to methylase activity 24,38 . However, the process involved in this offset mechanism remains elusive.

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The DNA hypermethylation of cloned embryos is one of the major failures of proper chromatin 1 8 5 remodeling during nuclear reprogramming 43,44 , however, in contrast to changes at a global level, 1 8 6 severe demethylation in important regions as satellite I and imprinted genes 43,44 could also be 1 8 7 responsible for such failure. Finally, its noteworthy that the unaltered transcript levels of UNC0638-treated embryos 1 8 9 suggest a very specific effect of the treatment on EHMT1/2 inhibition. This is consistent with the 1 9 0 finding that treatment with UNC0638 does not affect developmental rates as reported here and 1 9 1 previously in mice 45 , goats 46 , and sheep 27,47 . It is likely the effect of the UNC0638 is species-1 9 2 specific as only in pigs it was shown to improve development of SCNT-derived embryos 21,48 .

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Likewise, one might expect the UNC0638 treatment to improve developmental rates as it 1 9 4 significantly mitigated the levels of H3K9me3 in blastocysts; this mark is considered a main period to test H3K9me3 reduction-based approaches. Taken together, these data suggest that independently of their catalytic activity. Using a specific probe to inhibit the catalytic activity of EHMT1/2, we were able to H3K9me3, 5mC, and 5hmC levels independently of transcriptional changes. This suggests an 2 0 8 intense crosstalk among histone modifications and DNA methylation during nuclear 2 0 9 reprogramming in cattle. Nonetheless, although lower levels of H3K9me2 and H3K9me3 were 2 1 0 achieved at the blastocyst stage, EHMT1/2 inhibition did not increase developmental rates.

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Therefore, the aberrant epigenetic remodeling present in SCNT-derived embryos may not be 2 1 2 amended by inhibiting the catalytic activity of these epigenetic writers. All chemicals and reagents used were purchased from Sigma-Aldrich Chemical Company (St. Immunostaining of somatic cells. For H3K9me2 and H3K9me3 analysis, we procedure as buffer, a mouse antibody anti-H3K9me2 -Abcam (ab1220, 1:300) and a rabbit antibody anti- H3K9me3 -Abcam (ab8898, 1:500), overnight at 4 °C. Cells incubated without primary 2 5 1 antibodies were used as negative controls for all assays. In the next day, after washing 3x for 10 TransferTip; Eppendorf) was used to aspirate and remove the PB and metaphase II plates. The 3 1 2 aspirated cytoplasm was exposed to UV light to confirm for the presence of PB and metaphase 3 1 3 plate. After enucleation, one single donor cell from control or treatment, at the same cell passage, maturation, the fused SCNT couplets were activated with 5 µM ionomycin in TCM199-HEPES 3 2 0 (supplemented with 1 mg/mL fatty acid-free BSA) for 5 minutes and moved into TCM199- for the embryo treatment groups, zygotes from non-treated cells were split into drops of control mineral oil at 38.5°C and in atmosphere with 5% CO 2 . Developmental rates were evaluated at 3 2 8 day 2 for cleavage and day 7 for blastocyst. Control for oocyte quality, activation procedure, and 3 2 9 in vitro culture was made using activated metaphase II-arrested oocytes, using the same collected either for immunostaining procedure or for gene expression analysis. Embryo immunostaining. Immunostaining was performed as described previously 50 , with activation), and blastocyst at day 7 after activation were collected and washed in PBS+PVA.

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Next, RNA was treated with Deoxyribonuclease I, Amplification Grade (DNaseI, Invitrogen; 10 minutes, 37°C for 120 minutes and 85°C for 5 minutes. Quantitative real-time polymerase Mix (Applied Biosystems), 1 µM/mL of forward and reverse bovine-specific primers, 6.95ng of 3 7 7 cDNA and nuclease-free water. The primers were designed using the software Primer-BLAST 3 7 8 (NCBI) based upon sequences available in GenBank (Supplemental Table 1). The primers were 3 7 9 also sequenced to test their specificity. DNTM1, DNMT3A, DNMT3B and the reference genes geometric mean of PPIA and RPL15 reference genes. Software, San Diego, California, USA). Data were tested for normality of residuals and (developmental rates) were analyzed by Student's t-test. Differences with probabilities of P < Data Availability. The datasets generated during and/or analyzed during the current study are 3 9 8 available from the corresponding author on reasonable request. The authors would like to thank the staff and students at the LMMD, Jessica Brunhara Cruz, analysis, decision to publish, or preparation of the manuscript.      Proc. Natl. Acad. Sci. U. S. A. 111, 4139-44 (2014).     Biophys. Acta -Gene Regul. Mech. 1839, 169-177 (2014.    , characterization , and multipotentiality. Genet. Mol. Res. 14, 53-62 (2015).  Adult Mural Granulosa Cells. Biol. Reprod. 60, 996-1005 (1999).  replicates are shown. H3K9me2 levels were calculated in relation to total histone 3 (H3).

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Data as presented as mean ± S.E.M, and bars with different letters are significantly different respectively. All images were taken at the same magnification and at the same laser power, H3K9me2, H3K9me3, 5mC, and 5hmC levels in SCNT embryos derived from cells treated or