Identification Of Yap Alternative Splicing Isoforms And Their Roles In Embryonic Stem Cell

Embryonic stem cells(ESCs),which originate from the inner cell mass,can be propagated infinitely in a defined culture medium without losing pluripotency.For this reason,ESCs are an ideal model for understanding early stages of embryogenesis and attractive resources for regenerative medicine.Precisely characterizing gene functions and molecular mechanisms underlying the ESCs fate decision is therefore of great significance.ESCs fate is governed by the external and internal signals which regulate the balance between self-renewal and differentiation.The external stimuli are accepted and transduced by signal pathways to the transcriptional regulatory networks.High-throughput genomic and proteomic studies have revealed that the network is composed by three separated functional modules: the core(Oct4/Sox2/Nanog-centered,related to activate pluripotency genes and repress developmental genes),the Myc(c-Myc centered,mainly involved in regulating cellular metabolism,cell cycle,and protein synthesis)and the PRC(PRC1 and PRC2 complexes,contributed to repress lineage specific genes).Multiple signaling pathways,such as LIF/STAT3,BMP/Smad/Ids,Wnt/β-catenin and Mapk/Erk pathways are also known to be critical for ESCs fate determination.In contrast to these,the functions and signal integration mechanisms of Hippo pathway on regulating ESCs state are less well defined.The Hippo signaling cascade is highly conserved from Drosophila to mammals.A key effector of Hippo signaling is Yap,which regulates expression of target genes by binding to the transcription factors Teads(Tead1 to Tead4).The activated Hippo signaling sequentially activates MST1/2 and LATS1/2 kinase,which leads to serine phosphorylation of Yap and subsequent cytoplasmic retention and ubiquitin ligase-mediated proteasomal degradation.Mutation of the LATS1/2 phosphorylation sites mimics Hippo inhibition,constitutively activates Yap with ensuing nuclear entry,which in turn,causes evasion of cell contact inhibition and a proliferation advantage.Yap-null embryos die soon after gastrulation,whereas activated Yap causes organ overgrowth and aberrant tissue expansion in mice.However,although a large body of investigations has identified Yap as a pivotal switch in modulating cell proliferation and apoptosis,tissue regeneration and tumorigenesis,its functions in regulating pluripotency of ESCs still remain obscure and even contradictory;both necessary and dispensable for ESCs self-renewal have been reported.The Yap was first isolated as a Src family kinase Yes associated protein of 65 KDa.Over the past years,there have several splicing variants of Yap been isolated from the human,mouse and silkworm.Of note,the splicing events are remarkable conserved in all further amniotes up to human.The basic modular structure of Yap includes a TEAD factor-binding domain,one or two WW domain derived by differential mRNA splicing,a SH3 binding domain,a transcriptional activation domain(TAD)and a PDZ-binding motif(Fig.1a).Besides,a leucine zipper motif within the TAD domain can be preserved intact or disrupted by different alternative splicing(AS)events.These structural differences strongly suggest functional differences between splice isoforms of Yap.But to our knowledge,there are no reports regarding if and how Yap splicing products exist and play roles in ESCs fate decisions thus far.Here,based on the observation of the expression pattern of Yap splice variants,we respectively generated ESCs lines including Yap knockout(KO),Yap KO with single isoform rescue,over-expression(OE)of wild type(WT)or constitutively activated splice isoforms.Furthermore,we evaluated the functions of different Yap isoforms in regulating self-renewal maintenance,pluripotency exit,early and terminal differention of ESCs.The main concusions are summarized as below:1.Yap472 and Yap488 are universal present,and Yap splicing isoforms exhibit a heterogeneous expression pattern in ESCs;Yap472 and Yap488 were identified in ESCs cDNA samples by PCR and sequencing,and found to be widely expressed in different cells and tissues,but showing different expression patterns in single cells.Immunofluorescence staining also confirmed that Yap expression was heterogeneous in ESCs.2.Yap deletion decreases self-renewal efficiency,facilitates exit of pluripotency but blocks terminal differentiation of ESCs;By establishing Yap-KO cell lines,it was found that under self-renewal conditions,morphology of Yap-KO is flat and dispersed,and its proliferation efficiency is reduced,apoptosis is increased,cell G0 phase arrest,and the rate of Nanog positivity is decreased.Both qPCR and RNA-seq have found self-renewable genes down-regulation,and differentiation-related genes up-regulation;under differentiation conditions,qPCR found that early differentiation genes were up-regulated faster,and EB morphology showed that late differentiation was hindered.3.Yap472 and Yap488 function are redundant in maintaining ESCs identity and suitable early differentiation dynamics;The expression of Yap472 and Yap488 was restored in Yap-KO cells,respectively.Cell morphology,cell proliferation,apoptosis,clone formation experiments and qPCR detection of marker gene expression levels revealed that both Yap472 and Yap488 can restore damaged phenotype in Yap-KO.4.the functions of Yap splicing isoforms in regulating ESCs self-renewal are Teads dependent;CO-IP and HIP / HOP luciferase reporter experiments show that both Yap472 and Yap488 can bind to Teads and regulate downstream gene transcription levels.After inhibiting the binding of Tead to Yap,the Nanog positive rate was significantly reduced.5.c-Myc plays a major role in mediating the functions of Yap isoforms in ESCs self-renewal maintenance and exit;A cell line of inducible expression of c-Myc was established in Yap-KO cells.It was found that c-Myc can restore the morphology,damaged cell proliferation,cell cycle,apoptosis,clone formation ability,and Nanog positive rate of Yap-KO cells.It shows that both Yap472 and Yap488 can be combined with c-Myc.6.Yap472 is more essential than Yap488 for NE differentiation of ESCs.Yap-rescue cells were subjected to teratoma experiments,found obvious neural differentiation tendency in the teratomas of Yap472 rescue cells.RNA-seq analysis showed that most of the differential genes of Yap KO / R-472 cells were related to neural development.Three germ layer directional differentiation experiments found that Yap472 promotes NE development more than Yap488.7.Yap-472 OE and Yap-488 OE act similarly,albeit weakly,to promote self-renewal,delay(but not block)pluripotency exit and early differentiation of ESCs;After wild-type Yap OE,immunofluorescence experiment showed that most of Yap remained in the cytoplasm.Nanog positive rate,clone formation experiments,cell proliferation,cell cycle,and apoptosis experiments showed that OE Yap472 and Yap488 can promote self-renewal.The RNA expression level of marker genes was found to be slightly down-regulated by qPCR analysis.8.the constitutively active Yap impairs ESCs self-renewal and mainly induces TE trans-differentiation while the effect of Yap472-2SA is more pronounced.Mutation of Yap phosphorylation site achieved constitutive activation of Yap.After constitutively activating Yap,cell morphology,cell proliferation,and apoptotic rate tests showed that cell viability was significantly reduced.qPCR analysis and Cdx2 immunofluorescence experiments showed the increase of TE-labeled genes,particularly in Yap472-2SA OE cells.9.ectopic upregulation of Cdx2 appears the main reason why constitutively active Yap(mainly the Yap472)induces a loss of self-renewal and TE differentiation in ESCs.ChIP experiment showed that both constitutively activated Yap472 and Yap488 can bind to Cdx2.Cdx2 luciferase experiments show that Yap472 has a stronger ability to regulate downstream transcription than Yap488.After KD Cdx2 in constitutively activated Yap OE cells,apoptosis,and qPCR experiments showed that cell viability and cell differentiation phenotypes were largely restored.In conclusion,our findings reveal that the self-renewal/differentiation balance,Yap and the ESCs state interact as both cause and effect.Through buffering or escalating the pluripotency maintenance and development signals,the combined effects of different Yap splicing isoforms,expression and activation levels determine the self-renewal efficiency,differentiation kinetics and direction of ESCs.