Co-suppression Of PHLDA2 Expression By DNA Methylation And YY1 In Goat Placenta

Goat was one of the earliest domesticated animals as sheep.And goat breeding can be traced back to the Xia and Shang dynasty in china.Goat meat is not only a kind of food but also a kind of traditional Chinese medicine.Moreover,it is rich in nutrients,has excellent nourishing effects,and can prevent and alleviate many diseases.In 2019,compared with the previous year,the national output of meats reduced 10.2%,but the output of mutton increased by 2.6% in China.This shown that people's eating habits were being adjusted because the improvement of living standards.Goat meat is favored for more people with the high nutritional value and good teste.However,the low reproduction rate of goats has limited the goat breeding and market development.The placenta,one of the factors that affect reproduction performance,is a medium for nutrient supply between the mother and embryo.It is crucial for fetal development,growth and affects survival after birth.PHLDA2(pleckstrin homology-like domain,family A,member 2,also called as IPL or TSSC3),has the functions of regulating cell signaling,intra-package transportation and the like,is a maternally imprinted gene that expressed on the maternal allele in embryo and adult.A number of studies shown that PHLDA2 is a key gene for placental development.In embryos,the function loss of PHLDA2 lead to abnormal placental development,and the accumulation of glycogen storage.Furthermore,the overexpression of PHLDA2 inhibit fetal growth.Therefore,it is of great significance for uncover the regulate to PHLDA2 gene.According to previous experimental results: “PHLDA2 gene is highly expressed in goat placenta.”.Fetal tissues including the placenta were collected by Caesarean at 30,45,60,90 days post coitus(dpc).In order to investigate the different phase of placenta PHLDA2 gene expression,realtime PCR was performed to measure gene expression,simultaneously the expression profiles of PHLDA2 in different days post coitus were constructed in placenta,and Western blot was used to verify the results.we used the method of bisulfite sequencing PCR(BSP)and methylation-specific PCR(MSP)to draw methylation mapping from the promoterareas of PHLDA2 gene in the different days post coitus in placenta.Then,5-azacytidine(5-AzaC)was used to confirm the consequences in vitro.For further studied PHLDA2 molecular mechanism of gene expression,we constructed the luciferase report gene expression vectors about the 5' deletion promoter fragment,and analyzed promoter activity by transient transfection cell technology.This study achieved the following results:In order to confirm the expression of placenta in different days post coitus,real-time PCR results showed that mRNA levels of 45 dpc and 90 dpc were highest and lowest(P < 0.05),respectively.The Western blot analysis further supported the results of real-time PCR experiments.No CAAT-and TATA-like boxes was discovered in the upstream region and the first exon of the gene,but there was CpG is island between-621 and +379(island siza > 100,CG Percent > 50.0,Obs/Exp > 0.6).Potential transcription factor binding sites were predicted in the 5'flanking region.These transcription factoers include nuclear factor I-C(NIFC),the transcription factor ETS protooncogene(ETS1),ary1 hydrocarbon receptor(AHR)and Ying-yang 1(YY1).For further researched that the promoter upstream regulated the expression of PHLDA2,we constructed the luciferase report gene expression vectors that the 5'deletion fragments of goat PHLDA2 gene promoter were cloned and recombined into pGL3-Basic plasmids.Higher luciferase activity was observed in-420/+20bp compared to the pGL3-basic vector.And,-1023/+20bp show a 4-fold increase over-420/+20bp in luciferase activity(P < 0.05).Then,there were no significant alteration in promoter activity among-420/+20bp,-598/+20bp and-627/+20bp(P > 0.05).In summary,+20/-420 as a functional promoter and the strongest transcriptional activity of +20/-1023 in goat PHLDA2 promoter,we speculated that there are cis-acting or trans-acting element binding sites from-420 bp to-1023 bp.To detection whether DNA methylation was associated with the different days post coitus placenta tissue of goat PHLDA2 expression.As results,the lowest methylation level was detected at 45 days post coitus(14.2%).This suggests that promoter region methylation regulate PHLDA2 expression.To verify this conclusion,goat placenta trophoblast cells were treated with DNA methyl transferase inhibitor 5-AzaC and the hypermethylation of PHLDA2 promoter dual luciferase active vectors were constructed.As expected,5-AzaC treatment enhanced both PHLDA2 transcription and protein levels,and the hypermethylation of PHLDA2 promoter inhibited the activity of PHLDA2 promoter(P < 0.05).As there were one putative binding site for NFIC and AHR,and two supposed binding sites for YY1(called YY1-a and YY1-b)and EST1(called EST1-a and EST1-b)at the PHLDA2 promoter,the pGL3-1023 were mutated with site-directed mutagenesis technology.The luciferase activity of pGL3-1023-YY1-abMut significantly increased that compared with the pGL3-1023 vector(P < 0.05).,whereas the other mutants did not display any pivotal role in our dual-luciferase reporter assays(P > 0.05).To further verified the effect of YY1 on PHLDA2,YY1 siRNA and overexpression vectors were constructed and transfected in goat trophoblast cells.The results showed that the expression of PHLDA2 was decreased 0.5-fold in the YY1 overexpression compare with the siRNA cells.In the meantime,we noticed that the YY1-a binding site is located in the CpG island.In order to identify which YY1 site was more effective,we constructed the hypermethylation of luciferase report vectors by CpG methyltransferase(M.SssI).Compared with pGL3-1023,the luciferase activity of pGL3-1023 Me decreased significantly(P < 0.01).Moreover,compared with pGL3-1023 Me and pGL3-1023Me-YY1mut-b,the luciferase activity of pGL3-1023Me-YY1mut-a increased 3-fold and 1.5-fold,severally.This results proved that YY1 was more enriched at the YY1-a binding site.Meanwhile,the results of ChIP showed that YY1 was enriched in YY1-a more than YY1-b,which verified that the YY1 transcription factor bound in YY1-a in large quantities.Compared with NC siRNA,the results of ChIP showed that YY1 binding decreased after YY1 siRNA transfection,and the YY1 binding also decreased after 5-AzaC treatment.All results indicated that YY1 binding inhibits PHLDA2 expression at the YY1-a site and was related to promoter methylation.To verified HDACs family proteins interact with YY1,we used re-ChIP to examine.And the results demonstrated that YY1 interacts with HDAC1 and HDAC3 proteins to restrained PHLDA2 gene expression.In summary,the YY1 transcription factor prefers binding to CpG-methylation sequences,and inhibits goat PHLDA2 expression via recruiting HDAC1 and HDAC3.