Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

Inhibition of protein kinase C increases Prdm14 level to promote self-renewal of embryonic stem cells through reducing Suv39h-induced H3K9 methylation.

Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.

Rhox6 regulates the expression of distinct target genes to mediate mouse PGCLC formation and ESC self-renewal.

BACKGROUND: Mouse embryonic stem cells (mESCs) not only retain the property of self-renewal but also have the ability to develop into primordial germ cell-like cells (PGCLCs). However, knowledge about the mechanisms of transcriptional regulation is still limited. Rhox6, a member of the homeobox family that is located on the X chromosome, is highly expressed within PGCLCs in vivo and in vitro. However, the detailed effects of Rhox6 on PGCLC specification and mESC maintenance remain unclear. RESULTS: In this study, we found that overexpression of Rhox6 favors the formation of PGCLCs, while depletion of Rhox6 inhibits the generation of PGCLCs. Mechanistically, Rhox6 directly induces the expression of Nanos3 during the specification of PGCLCs. Subsequently, downregulation of Nanos3 expression is sufficient to decrease the ability of Rhox6 to induce PGCLC formation. Moreover, we found that depletion of Rhox6 expression facilitates the self-renewal of mESCs. High-throughput sequencing revealed that suppression of Rhox6 transcription significantly increases the expression of pluripotency genes. Functional studies further demonstrated that Rhox6 directly represses the transcription of Tbx3. Therefore, knockdown of the expression of the latter impairs the self-renewal of mESCs promoted by Rhox6 downregulation. CONCLUSIONS: Our study reveals that overexpression of Rhox6 is beneficial for PGCLC generation through induction of Nanos3, while downregulation of Rhox6 contributes to mESC self-renewal by increasing Tbx3. These findings help elucidate the early development of mouse embryos.

Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity.

Prdm14 plays an important role in the maintenance of mouse embryonic stem cell (mESC) pluripotency and the specification of primordial germ cells (PGCs). However, the mechanism downstream of Prdm14 is still not fully understood. Here, using high-throughput sequencing, chromatin immunoprecipitation, and luciferase reporter assays, we show that Prdm14 directly binds to the promoter of Socs3 and represses its transcription to increase the phosphorylation level of Stat3 protein, a critical downstream effector of LIF. Therefore, ectopic expression of Socs3 is able to decrease the ability of Prdm14 to promote mouse mESC self-renewal and PGC-like cell generation. As expected, similar phenotypes were observed in Prdm14-transfected mESCs after knockdown of Stat3 transcripts or treatment with a pan-inhibitor of JAKs, positive modulators of the LIF/Stat3 signaling pathway. These data will facilitate a better understanding of the regulatory network governing ESC identity and germ cell development.

MK2 promotes Tfcp2l1 degradation via ß-TrCP ubiquitin ligase to regulate mouse embryonic stem cell self-renewal.

Tfcp2l1 can maintain mouse embryonic stem cell (mESC) self-renewal. However, it remains unknown how Tfcp2l1 protein stability is regulated. Here, we demonstrate that ß-transducin repeat-containing protein (ß-TrCP) targets Tfcp2l1 for ubiquitination and degradation in a mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-dependent manner. Specifically, ß-TrCP1 and ß-TrCP2 recognize and ubiquitylate Tfcp2l1 through the canonical ß-TrCP-binding motif DSGDNS, in which the serine residues have been phosphorylated by MK2. Point mutation of serine-to-alanine residues reduces ß-TrCP-mediated ubiquitylation and enhances the ability of Tfcp2l1 to promote mESC self-renewal while repressing the speciation of the endoderm, mesoderm, and trophectoderm. Similarly, inhibition of MK2 reduces the association of Tfcp2l1 with ß-TrCP1 and increases the self-renewal-promoting effects of Tfcp2l1, whereas overexpression of MK2 or ß-TrCP genes decreases Tfcp2l1 protein levels and induces mESC differentiation. Collectively, our study reveals a posttranslational modification of Tfcp2l1 that will expand our understanding of the regulatory network of stem cell pluripotency.

Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells.

Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small-molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK prodifferentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of an MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency.

Gadd45g initiates embryonic stem cell differentiation and inhibits breast cell carcinogenesis.

Many self-renewal-promoting factors of embryonic stem cells (ESCs) have been implicated in carcinogenesis, while little known about the genes that direct ESCs exit from pluripotency and regulate tumor development. Here, we show that the transcripts of Gadd45 family genes, including Gadd45a, Gadd45b, and Gadd45g, are gradually increased upon mouse ESC differentiation. Upregulation of Gadd45 members decreases cell proliferation and induces endodermal and trophectodermal lineages. In contrast, knockdown of Gadd45 genes can delay mouse ESC differentiation. Mechanistic studies reveal that Gadd45g activates MAPK signaling by increasing expression levels of the positive modulators of this pathway, such as Csf1r, Igf2, and Fgfr3. Therefore, inhibition of MAPK signaling with a MEK specific inhibitor is capable of eliminating the differentiation phenotype caused by Gadd45g upregulation. Meanwhile, GADD45G functions as a suppressor in human breast cancers. Enforced expression of GADD45G significantly inhibits tumor formation and breast cancer metastasis in mice through limitation of the propagation and invasion of breast cancer cells. These results not only expand our understanding of the regulatory network of ESCs, but also help people better treatment of cancers by manipulating the prodifferentiation candidates.

The transcription factor Tfcp2l1 promotes primordial germ cell-like cell specification of pluripotent stem cells.

Primordial germ cells (PGCs) are common ancestors of all germline cells. However, mechanistic understanding of how PGC specification occurs is limited. Here, we identified transcription factor CP2-like 1 (Tfcp2l1), an important pluripotency factor, as a pivotal factor for PGC-like cell (PGCLC) specification. High-throughput sequencing and quantitative real-time PCR analysis showed that Tfcp2l1 expression is gradually increased during mouse and human epiblast differentiation into PGCLCs in vivo and in vitro. Consequently, overexpression of Tfcp2l1 can enhance the specification efficiency even without inductive cytokines in mouse epiblast-like cells derived from embryonic stem cells, while knockdown of Tfcp2l1 significantly inhibits PGCLC generation. Mechanistic studies revealed that Tfcp2l1 exerts its function partially through the direct induction of PR domain zinc finger protein 14, a key PGC marker, as downregulation of the PR domain zinc finger protein 14 transcript can impair the ability of Tfcp2l1 to direct PGCLC commitment. Importantly, we finally demonstrated that the crucial role of the human homolog Tfcp2l1 in promoting PGCLC specification is conserved in human pluripotent stem cells. Together, our data uncover a novel function of Tfcp2l1 in PGCLC fate determination and facilitate a better understanding of germ cell development.

Inhibition of MTA2 and MTA3 induces mesendoderm specification of human embryonic stem cells.

Fully understanding the regulatory network under the pluripotency of embryonic stem cells (ESC) is a prerequisite for their safe application. Here, we addressed the characteristics of metastasis-associated (MTA) family members in human ESCs and found that knockdown of the expression of MTA2 and MTA3, but not MTA1, would induce differentiation. High-throughput sequence and quantitative real-time PCR showed that the decreased MTA2 or MTA3 gene transcript mainly led to the emergence of mesendoderm associated markers. Finally, based on the chemical small molecule library screening, we observed that addition of ID8, a specific inhibitor of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), was able to impair the differentiation phenotype induced by MTA2 and MTA3 reduction. Functional assay showed that ID8 could mediate differentiation caused by MTA2 or MTA3 knockdown mainly through inhibition of DYRK4 activity. Therefore, our finding provides the evidence that the functions of MTA family genes in human ESCs are different. Revealing the function of MTA in ESCs with different pluripotency states will help us better understand and apply stem cells.

Inhibition of protein kinase D by CID755673 promotes maintenance of the pluripotency of embryonic stem cells.

The identification of novel mechanisms to maintain embryonic stem cell (ESC) pluripotency is of crucial importance, because the currently used culture conditions are not suitable for ESCs from all species. In this study, we show that the protein kinase D (PKD) inhibitor CID755673 (CID) is able to maintain the undifferentiated state of mouse ESCs in combination with the mitogen-activated protein kinase kinase (MEK) inhibitor. The expression levels of PKD members, including PKD1, PKD2 and PKD3, were low in mouse ESCs but significantly increased under differentiation conditions. Therefore, depletion of three PKD genes was able to phenocopy PKD inhibition. Mechanistically, PKD inhibition activated PI3K/AKT signaling by increasing the level of AKT phosphorylation, and the addition of a PI3K/AKT signaling pathway inhibitor partially reduced the cellular response to PKD inhibition. Importantly, the self-renewal-promoting effect of CID was maintained in human ESCs. Simultaneous knockdown of the three human PKD isoforms enabled short-term self-renewal in human ESCs, whereas PI3K/AKT signaling inhibition eliminated this self-renewal ability downstream of the PKD inhibitor. These findings expand our understanding of the gene regulatory network of ESC pluripotency.

ß-catenin stimulates Tcf7l1 degradation through recruitment of casein kinase 2 in mouse embryonic stem cells.

Activation of the Wnt/ß-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2ß, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/ß-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/ß-catenin signaling pathway in ESCs.

The transcription factor TFCP2L1 induces expression of distinct target genes and promotes self-renewal of mouse and human embryonic stem cells.

TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. However, the direct TFCP2L1 target genes are uncharacterized. Here, using gene overexpression, immunoblotting, quantitative real-time PCR, ChIP, and reporter gene assays, we show that TFCP2L1 primarily induces estrogen-related receptor ß (Esrrb) expression that supports mouse ESC identity and also selectively enhances Kruppel-like factor 4 (Klf4) expression and thereby promotes human ESC self-renewal. Specifically, we found that in mouse ESCs, TFCP2L1 binds directly to the Esrrb gene promoter and regulates its transcription. Esrrb knockdown impaired Tfcp2l1's ability to induce interleukin 6 family cytokine (leukemia inhibitory factor)-independent ESC self-renewal and to reprogram epiblast stem cells to naïve pluripotency. Conversely, Esrrb overexpression blocked differentiation induced by Tfcp2l1 down-regulation. Moreover, we identified Klf4 as a direct TFCP2L1 target in human ESCs, bypassing the requirement for activin A and basic fibroblast growth factor in short-term human ESC self-renewal. Enforced Klf4 expression recapitulated the self-renewal-promoting effect of Tfcp2l1, whereas Klf4 knockdown eliminated these effects and caused loss of colony-forming capability. These findings indicate that TFCP2L1 functions differently in naïve and primed pluripotency, insights that may help elucidate the different states of pluripotency.

Comprehensive integrated analysis of gene expression datasets identifies key anti-cancer targets in different stages of breast cancer.

Breast cancer is one of the primary threats to women's health worldwide. However, the molecular mechanisms underlying the development of breast cancer remain to be fully elucidated. The present study aimed to investigate specific target gene expression profiles in breast cancer tissues in general and in different breast cancer stages, as well as to explore their functions in tumor development. For integrated analysis, a total of 5 gene expression profiling datasets for 3 different stages of breast cancer (stages I-III) were downloaded from the Gene Expression Omnibus of the National Center for Biotechnology Information. Pre-processing of these datasets was performed using the Robust Multi-array Average algorithm and global renormalization was performed for all studies. Differentially expressed genes between breast cancer patients and controls were estimated using the empirical Bayes algorithm. The Database for Annotation, Visualization and Integrated Discovery web server was used for analyzing the enrichment of the differentially expressed genes in Gene Ontology terms of the category biological process and in Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, breast cancer target genes were downloaded from the Thomson Reuters Integrity Database. We merged these target genes with the genes in breast cancer datasets. Analysis of anti-breast cancer gene networks was performed using the Genome-scale Integrated Analysis of Gene Networks in Tissues web server. The results demonstrated that the normal functions of the cell cycle, cell migration and cell adhesion were altered in all stages of breast cancer. Furthermore, 12 anti-breast cancer genes were identified to be dysregulated in at least one of the three stages. Among all of these genes, ribonucleotide reductase regulatory subunit M2 (RRM2) exhibited the highest degree of interaction with other interacting genes. Analysis of the network interactions revealed that the transcription factor of RRM2 is crucial for cancer development. Other genes, including mucin 1, progesterone receptor and cyclin-dependent kinase 5 regulatory subunit associated protein 3, also exhibited a high degree of interaction with the associated genes. In conclusion, several key anti-breast cancer genes identified in the present study are mainly associated with the regulation of the cell cycle, cell migration, cell adhesion and other cancer-associated cell functions, particularly RRM2.

Modulation of STAT3 phosphorylation by PTPN2 inhibits naïve pluripotency of embryonic stem cells.

STAT3 phosphorylation at tyrosine 705 (STAT3pY705 ), triggered by the addition of the leukemia inhibitory factor (LIF), can maintain mouse embryonic stem cell (mESC) self-renewal and reprogram mouse epiblast stem cells (EpiSCs) to enter a naïve pluripotent state. The activation of STAT3pY705 occurs mainly through Janus kinases. However, it remains unclear how STAT3pY705 levels are decreased in mESCs. Our study shows that upregulation of the protein tyrosine phosphatase (PTPN2) inhibits STAT3 activity by reducing its phosphorylation level and promotes mESC differentiation, whereas PTPN2 knockout by CRISPR/CAS9 delays mESC differentiation. Consistently, PTPN2 knockdown facilitates the generation of mESC-like colonies in STAT3-overexpressing EpiSCs. PTPN2-mediated STAT3 activity, thus, contributes to the exit of ESCs from the pluripotent ground state. These findings expand the current understanding of the regulatory network of naïve pluripotency.