| Mouse embryonic stem cells(mESCs),which are derived from the inner cell mass(ICM)of blastocyst stage embryos,have indefinite self-renewal ability and multi-lineage differentiation capacity.These two unique properties make ESCs attractive and accessible models to study early embryogenesis and developmental process.In particular,previous studies have unveiled the key roles of extrinsic signals and interconnected transcription networks in regulating ESC pluripotency.Extrinsically,leukemia inhibitory factor(LIF)/Stat3 and Wnt/β-catenin pathways are pivotal to sustain ESC self-renewal,whereas Fgf4/Mek/Erk pathway is important for ESCs to initiate differentiation.Intrinsically,the core transcription factors(Pou5f1,Sox2,and Nanog)act together with other factors(e.g.,Esrrb,Klf2,Klf4,Klf5,Nr5a2,Rex1,and Myc)and cooperate with featured epigenetic modifications(e.g.,H3K4me3/H3K27me3 bivalent domains related to developmental genes)to form a transcription regulatory network that consolidates ESC identity.Moreover,the roles of post-transcriptional processes(e.g.,m RNA splicing),cellular mechanics,phase separation and ionic homeostasis in regulating ESC pluripotency have also been revealed by recent studies.Understanding molecular mechanisms that underpin pluripotency maintenance not only provides insights to cell fate commitment during early embryonic stages,but also facilitates advancement in the field of regenerative medicine,organoid culture,and drug discovery.Traditionally,ESCs can be propagated indefinitely in the presence of LIF and serum(SL)in vitro and are referred as “conventional” ESCs.They are not in a singular developmental state,but rather contain multiple sub-populations from naive to formative and primed pluripotency states,showing heterogeneity and metastability.Recently,using a glycogen synthase kinase-3 inhibitor(Gsk3i)and a mitogen-activated kinase kinase inhibitor(Meki)in combination with LIF(2iL)has established the ground state of pluripotency.Gsk3 i elevates Wnt/β-catenin signaling and strengthens the derivation and proliferation of undifferentiated ESCs,while Meki represses Mek/Erk signaling and prevents differentiation.Previous studies have demonstrated remarkable differences between SL and 2iL ESCs.Transcriptome analysis has uncovered that several pluripotency factors(e.g.,Nanog,Rex1,and Prdm14)are heterogeneously expressed,and some lineage-specific genes(e.g.,Fgf5,Otx2,and Dnmt3b)are actively transcribed in SL ESCs.In contrast,2iL ESCs display homogeneous expression of Nanog,Rex1 and Prdm14,as well as negligible levels of lineage-specific genes.More interestingly,for epigenetic landscapes,2iL ESCs show reduced number of bivalent regions,global hypo-methylation,and increased chromatin accessibility,compared with SL ESCs.Furthermore,it is generally acknowledged that 2iL and SL ESCs correspond to different developmental stages in vivo.2iL ESCs closely resemble embryonic day(E)3.5-E4.5 ICM in pre-implantation embryos,whereas SL ESCs may represent E5.5 epiblast(Epi)of early post-implantation stage.Therefore,2iL ESCs are representative of a more robust pluripotency state,while conventional SL condition primes ESCs for germ-layer differentiation.Although multi-omic data by far has comprehensively revealed molecular phenotype differences between ESCs under metastable and ground states,only a few genes(e.g.,Dax1,Prdm15,and Myc)of their functions are compared in different states of pluripotency.Little is known about functional genes that play general and state-specific roles in regulating ESC self-renewal of different states of pluripotency.Genome-wide functional screen is a systematical and powerful tool to find out unappreciated gene functions in large-scale.Recently,CRISPR/Cas9 system,due to its high editing efficiency and low off-targeting effects compared to RNA interference(RNAi),was widely and successfully adopted in screening for essential genes and drug targets in various cell types.Here,we performed parallel CRISPR/Cas9-KO screens in SL and 2iL ESCs to identify and compare self-renewal regulators in different pluripotency states.Additionally,we verified the novel roles of two candidates(Zfp598 and Zfp296)in regulating self-renewal of ESCs under different states of pluripotency.Zfp598 was required for both SL and 2iL ESC self-renewal,while Zfp296 was a positive and negative regulator for SL and 2iL ESC self-renewal,respectively.Our data might present resources for further exploration of cell fate regulation.Below are our main conclusions:1.There were 2264 self-renewal-promoting(Pro-s)genes and 48 self-renewalinhibiting(Inh-s)genes in SL screen,and 612 Pro-s genes and 67 Inh-s genes in 2iL screen.2.The self-renewal regulatory genes in SL and 2iL mESCs could be divided into two categories,i.e.,general and state-specific self-renewal regulators.They could be further divided into seven groups.To be more specific,general self-renewal regulators included(Ⅰ)517 “shared Pro-s” and(Ⅱ)3 “shared Inh-s”,which were common in SL and 2iL mESCs.State-specific self-renewal regulators included(Ⅲ)1728 “SL special Pro-s” and(Ⅳ)45“SL special Inh-s” that were only visible in the SL screen;(Ⅴ)95 “2iL special Pro-s” and(Ⅵ)45 “2iL special Inh-s” that were only detected in the 2iL screen;(Ⅶ)19 “SL Pro-s and2 i L Inh-s” that showed opposite changes in SL and 2iL screens.3.“Shared Pro-s” genes were highly expressed in the PSCs of undifferentiated status,while their expression levels fell upon PSC differentiation.And these “shared Pro-s” genes mainly were involved in the basic,essential cellular processes,like cell cycle regulation,translation,transcription,and RNA processing.4.Zfp598 was important for both SL and 2iL mESC self-renewal.Zfp598 knockout(KO)comprised the growth and colony formation ability of both SL and 2iL mESCs.Under SL condition,Zfp598 KO led to more loose and flattened colonies that were representative of differentiated ESCs.While under 2iL condition,although the morphology of Zfp598 KO mESC was almost unchanged,their sizes became smaller.Additionally,Zfp598 KO led to significant downregulation of pluripotency-associated genes and upregulation of lineage-specifying genes.5.In SL condition,the expression levels of “SL special Pro-s” genes were higher than those of “2iL special Pro-s” genes.On the contrary,in 2iL condition,the expression levels of “2iL special Pro-s” genes were higher than those of “SL special Pro-s” genes.6.“2iL special Pro-s” genes were more enriched for lipid metabolism related processes than “SL special Pro-s” genes.7.The expression levels of “SL special Inh-s” genes and “2iL special Inh-s” genes were upregulated during the course of mESC differentiation.8.The protein products of “SL Pro-s and 2iL Inh-s” genes mainly contained upstream regulators of Hippo and m TORC signaling pathways,as well as chromatin regulators and transcription regulators.9.Zfp296 positively and negatively regulated the self-renewal of SL and 2iL mESCs,respectively.Under SL condition,Zfp296 KO led to impaired mESC growth and colony formation capacity.Zfp296 KO mESC showed loose and flattened morphology,with decreased AP staining activity.Additionally,Zfp296 KO downregulated the expression levels of pluripotency-associated genes,while upregulated those of lineage-specifying genes.On the contrary,under 2iL condition,Zfp296 KO promoted mESC growth and colony formation ability.And Zfp296 KO increased the expression level of Pou5f1,while didn’t influence that of lineage-specific genes.In summary,we have successfully performed parallel genome-scale CRISPR/Cas9loss-of-function screenings in SL and 2iL mESCs to identify and compare self-renewal genes under distinct states of pluripotency.Our results might present resources for further studies of cell fate regulation. |
