Reprogramming Dynamics Of Histone H3Lysine27Acetylation During Pre-implantation Development Of Pig Somatic Cloned Embryos

Somatic cloning or somatic cell nuclear transfer (SCNT) provides a possibility formaking transgenic pigs with novel traits of breeding value in disease resistance, qualityimprovement, grain saving and environment protection. SCNT is of great help to creatgenetic modified pigs as human disease models, establish porcine mammary glandbioreactors to produce pharmaceutical proteins, provide donor sources of human organxenotransplantation, and present revolutionary means of biological basic researches, whencombined in particular with transgenesis and stem cell biotechnologies. Unfortunately, theefficiency of SCNT is still extremely low so far, and incomplete epigenetic reprogrammingof somatic cells is believed to be the major causes of poor efficiency. DNAmethylation/demethylation,histone modifications (acetylation/deacetylation,methylation/demethylation, SUMOylation, ubiqutination, and phosphorylation etc.) aretwo major epigenetic modification events. As the core of the nucleosome in chromosomesof eucaryotic cells, the histone may affect gene expression of the corresponding regionwhen the physical and chemical changes happened, which is involved in the regulation ofeukaryotic cells growth and development. However, studies in reprogramming of histonemodifications during SCNT are scarce, especially whether histone acetylation modificationis involved in pre-implantation development of pig embryos is unknown. In the presentstudy, we detected the H3K27ac level at the different stages in in vitro fertilizationembryos (IVFE), nuclear transfer embryos (NTE), parthenogenetic activation embryos(PAE) during pre-implantation development in pigs in order to reveal spatial and temporalexpression profiles of H3K27ac during pre-implantation development of pig embryos, andthis would not only be helpful to find the correlation of the H3K27ac expression and thedevelopment fate of embryos from different origins, but also beneficial to future revealingof regulatory roles that H3K27ac played during pre-implantation development of pigembryos.Experiment I was conducted to investigate spatial and temporal expression pattern ofH3K27ac during pre-implantation development of pig embryos. Firstly, peptidecompetition assay was performed in order to examine specificity of antibody againstH3K27ac. The antibody against H3K27ac was pre-incubated with the peptide prior to useindirect immunofluorescence assays of pig PA blastocysts. The results showed that PAblastocysts had no H3K27ac signal, and indicated that the antibody against H3K27ac wasspecific. Then we examined the distribution pattern of the H3K27ac in NTE and PAE at15 min，30min and1hour after the electrical activation. According to the H3K27ac level ofNTE for1h after the electrical activation, we then tracked the source of the H3K27acmodification. The H3K27ac levels in NTE was analysed at different stages (2hpa,6hpa,12hpa,14hpa,16hpa,1cell,2cell,4cell,8cell, morula, EB, EXPB and HB stages), while inIVFE we used embryos at6hpi,8hpi,12hpi,16hpi,8hpi,1cell,2cell,4cell,8cell, morula,EB, EXPB and HB stages for comparison. The results showed that: the H3K27ac level ofMII stage oocytes was very low, and this level was not increased in oocytes until1h afterthe electrical activation. The H3K27ac level of NTE donor cell lines was very high, similarto that in NTE at15min,30min and1hour after the electrical activation. In NTE, theH3K27ac at2hpa,6hpa,12hpa showed quite high level until14hpa, but start to decrease at16hpa, and to the minimum at4cell and8cell stages, but come back to high level at morulastage, and to the maximum at blastocyst stage. This distribution pattern of H3K27ac inNTE is the same to that in PAE and IVFE. However, the H3K27ac level at HB in NTEdecrease as happened at HB in PAE, while H3K27ac at HB in IVFE still at high level.Results indicated that the H3K27ac expression level in NTE reflects its memory from thedonor cells, and the active removal of H3K27ac at4cell and8cell stages may be moreconducive to embryonic genome activation.In Experiment II, we tested wether H3K27ac falling-off at4cell and8cell stage pigembryos was dependent on DNA replication, RNA transciption and protein translationbefore embryonic genome activation. The oocytes,6hours after parthenogenetic activation,were treated by3μg.mL-1aphidicolin for12hours,25μg.mL-1α-amanitin for24hours,25μg.mL-1cycloheximide for24hours, respectively, and such inhibitions were thenterminated and cultured in vitro for24hours,12hours and12hours respectively withPZM-3. Results showed that the H3K27ac level in the6hpa oocytes treated by aphidicolin,α-amanitin and cycloheximide was very low, and similar to the control group, but theH3K27ac level in6hpa oocytes (without inhibition) was quite high. Data indicated that theremoval of H3K27ac may be an active process.Experiment III was to explore the dynamic changes of acetyltransferases anddeacetyltransferases which potentially regulate H3K27ac. The RNA was extracted fromGV stage (n=20) and MII stage oocytes (n=20), and from IVFE, NTE and PAE at stages of1cell,2cell,4cell and blastocyst (n=20each stage, each origin). Quantitative real-time PCRresults showed that: HACD1, HACD2, CBP and MBD3expression level at1cell,2cell,4cell and blastocyst stages IVFE are higher than those at same stages NTE and PAE.HACD2in NTE was highly expressed, which are higher at4cell than at1cell，2cell and blastocyst. All this implies that more transcription, translation and histonedeacetyltransferases synthesis happened, making the effective removal of H3K27acetylation at4cell and8cell stagesmore conducive to embryonic genome activation.In Experiment IV, we attemped to analyze molecular mechanism involving inefficiency improvement of NTE treated by trichostatin (TSA). After electrical activation,the cloned embryos were treated with50nM TSA for24hours, then cultured after beingwashed with fresh PZM-3to remove TSA, and those embryo without treatment serves ascontrol group. The cleavage and blastocyst formation on NTE was observed and recordedon the2day and7day respectively, and the blastocysts were collected for indirectimmunofluorescence. Results showed that the TSA treatment significantly increased theblastocyst rate (42.5%±2.8%vs.16.0%±3.7%) and the total number of cells of theblastocysts (77.4±4.9vs.54.0±7.6). The H3K27ac level of the treatment group was closerto that of IVF blastocysts compared with the control group. Results indicated that the TSAcould improve NTE development efficiency and improve H3K27ac level of NTE atblastocyst stage.In all, the current study is the first, to our knowledge, to explore the histoneH3K27ac genome-wide expression patterns during pre-implantation development of pigembryos. Our data not only revealed the spatial and temporal expression pattern ofH3K27ac in pig embryos, but also showed differences exist in different types of embryos.Moreover, we found that the removal of H3K27ac before embryonic genome activationmay be an active process, and this active process seemed to be more conducive toembryonic genome activation. Our result then implies that the H3K27ac may not only beinvolved in regulating development of pig pre-implantation embryos, but also beresponsible to some extent for NTE poor fate. All this laid a solid foundation for futureoptimization of somatic reprogramming NTE in pigs.