Molecular Mechanism Study Of Histone Modulator CARM1Regulates Pluripotency Of Human Embryonic Stem Cells

Background and objectiveHuman embryonic stem cells(hESC) are valuable resources for clinical therapies,biology and pharmacology research because of their pluripotency and unlimitedself-renewal ability.The pluripotency and self-renewal properties are regulated by anarray of transcription factors:Oct4, Nanog and Sox2.However, except the well-knownpathways of IGF, FGF and TGFβ, relatively little is known about their upstreamsignals,especially of epigenetic regulation pathways. Recent studies indicate that histoneH3methylation at Arg-17(R17) and Arg-26(R26), catalyzed by coactivator-associatedprotein-arginine-methyltransferase1(CARM1), predispose blastomeres to contributetothe pluripotent cells of the inner cell mass (ICM) in mouse embryo by elevating theexpression level of Nanog.Histone modifications are pivotal during hESC self-renewal and differentiation.However, while histone lysine methylations and their modulators have been extensivelyinvestigated in the maintenance of human ES cell self-renewal, pluripotency and somaticcell reprogramming; there exists little complementary information for the role of histonearginine methylations in the pluripotency regulation. As one of the histone argininemethyltransferases, CARM1catalyzes methylation at H3R17and R26in the N-terminalregion and at one or more of four arginines (128/129/131/134) at the C-terminus.As totranscriptional regulation; the H3R17and H3R26are major activation mark. Recently, aCARM1specific inhibitor was found which can be further used in loss of function studiesMicroRNAs (miRNAs), which are evolutionarily conserved noncoding RNAs withlengths of18-24nucleotides, are proved to play critical roles in the regulation of geneexpression and multiple cellular processes. Through base pairing with mRNAs atpartially or fully complementary sites, miRNAs induce mRNA cleavage or translationalrepression. The cell type-specific expression signature of miRNAs in ESCs has been usedsuccessfully to distinguish ESCs from differentiated cell types. More than100miRNAsare differentially expressed in hESC and the differentiated embryoid bodies.Our findings indicated that CARM1not only up-regulats Nanog in mouse embryo,but also in hESC. By using the bioinformatics tool,12candidate miRNAs were selectedand we further found miR-181family members regulate the expression of CARM1via directly targeting its3’untranslated regions (3’UTR). Taken together, our study identifiedCARM1plays an essential role in maintaining pluripotency of hESCs and reveals a directlink between histone modulator and posttranscriptional regulator in hESCs self-renewingprocess.Part Ⅰ The effect of CARM1on the pluripotency ofmouse embryo and hESCMethods:(1) Synthesize CARM1mRNA in vitro and construct the pcDNA3.1-flag－-CARM1recombinant plasmids.: CARM1mRNA was transcribed in vitro usingmMessage mMachine T7Ultra kit (Ambion) from PCR-product template encodingCARM1. The complementary DNA encoding CARM1lacks its3’UTR wasPCR-amplified from human genomic DNA and subcloned into the pcDNA3.1-flag－vector to construct the pcDNA3.1-flag－-CARM1recombinant plasmid.(2) Construct andanalysis CARM1overexpression (OE) models of the early mouse embryo/hESC andCARM1knock down (KD) model of hESC: Collect mouse embryos in the2-cell stageand inject the CARM1mRNA into one of the blastomere. pcDNA3.1-flag－-CARM1recombinant plasmids were transfected into hESC using Lipofectamine2000kit. ThesiRNAs specifically targeting CARM1were synthesized by GenePharma and transfectedinto hESC. Immunofluorescence was performed to detect the expression of Nanog inmouse embryo. Realtime-PCR and Westernblot were performed to analysis theexpression transcription factors (TF) and marker genes for specific differentiated lineages.The pluripotent morphology was examined by alkaline phosphatase (AP) staining.(3)The effect of CARM1in differentiated hESC: Differentiation was induced in hESCmedium deprived of bFGF. The pluripotent morphology was examined under bright field.Results:(1) The polyA tail of CARM1mRNA was successfully added. pcDNA3.1-flag－-CARM1recombinant plasmids passed sequencing (2) The expression of Nanog increasedin CARM1OE mouse embryo/hESC and decreased in CARM1KD hESC. Theexpression of marker genes for specific differentiated lineages increased in in CARM1KD hESC. CARM1KD hESC formed a lower proportion of AP-positive colonies than incontrol cells.(3) CARM1OE hESC maintained the normal morphologies for as long as2days in contrast to wild-types.Conclusions:(1) CARM1mRNA and recombinantplasmids were successfully constructed.(2) CARM1elevates the expression of Nanog in the early mouse embryo and hESC.(3) CARM1maintains the pluripotency of hESC andleads hESCs to resist differentiation.Part ⅡThe mechanism of CARM1in maintaining pluripotency of hESCMethods: To investigate whether CARM1directly targets pluripotency genes Oct4,Sox2and Nanog, we performed ChIP analysis on undifferentiated hESCs, CARM1KDhESCs and CARM1OE hESCs. Results: CARM1and histone H3R17dimethylationsignificantly enriched at the promoter of Oct4and Sox2in both undifferentiated hESCsand CARM1OE hESCs whereas clearly decreased in CARM1KD hESCs. Conclusions:CARM1directly catalyze histone H3arginine methylation of the promoter of Oct4andSox2, the elevated CARM1levels lead to CARM1recruitment at the Nanog promoter.Part Ⅲ miR-181c represses pluripotency via targets CARM1in hESCMethods:(1) The expression changes of CARM1and H3R17dimethylation indifferentiated hESC: Realtime-PCR and Westernblot were performed to analysis theexpression of CARM1and H3R17dimethylation.(2) Identify the miRNAs directly targetCARM1: Analyzed the expression of12miRNAs with the highest context score byTargetScan in undifferentiated hESCs and differentiated hESCs. Verify the candidatemiRNAs in Dual-Luciferase Reporter Assay System.(3) The effect of overexpressionmiR-181in hESC: miR-181c mimics were synthesized by GenePharma and transfectedinto hESC. Realtime-PCR and Westernblot were performed to analysis the expression ofTF and marker genes for specific differentiated lineages. The pluripotent morphology wasexamined by AP staining.(4) CARM1rescue constructs inhibits the effect ofoverexpression of miR-181c: Cotransfected miR-181c mimics and CARM1RNAi-resistant expression vector： pcDNA3.1-flag－-CARM1， Realtime-PCR wasperformed to analysis the expression of TF，the pluripotent morphology was examined byAP staining. Results:(1) CARM1and H3R17dimethylation was down-regulated indifferentiated hESC after removing of bFGF.(2) miR-181family members wereup-regulated in differentiated hESC, miR-181a/b/c/d directly target CARM13’UTR (3)Overexpression of miR-181c down-regulated CARM1, Oct4, Nanog andSox2.,up-regulated differentiation genes. Overexpression of miR-181c formed a lowerproportion of AP-positive colonies than in control cells.(4) Overexpression CARM1 rescue constructs restored the expression of Nanog and the proportion of AP-positivecolonies partially. Conclusions:(1) CARM1and H3R17dimethylation isdown-regulated in differentiated hESC.(2) miR-181directly target CARM1.(3)miR-181c impairs the pluripotency of hESC by targeting CARM1.(4)CARM1restoresthe pluripotency of hESC to a certain extent.Conclusions60In summary, the work presented here elucidates a direct link between histonemodulator and posttranscriptional regulator, reveals important insights into the role ofCARM1/H3arginine methylation in the maintenance of hESC self-renewal and suggestsfor the first time, how miR-181family downstream target epigenetic factors reconfigurethe H3arginine methylation signature during the process of hESC differentiation. Ourfindings thereby offer a unique perspective for understanding the mechanisms ofpluripotency maintaining of ESCs in the human.