Chaperone-mediated autophagy regulates the pluripotency of embryonic stem cells.
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ABSTRACT:
SUBMITTER: Xu Y
PROVIDER: S-EPMC7939502 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transcription factor EB (TFEB), a well-known master regulator of autophagy and lysosomal biogenesis, is a member of the microphthalmia family of transcription factors (MiT family). Over the years, TFEB has been shown to have diverse roles in various physiological processes such as clearance for intracellular pathogenic factors and having developmental functions such as dendritic maturation, as well as osteoclast, and endoderm differentiation. However, in the present study, we propose a novel mechanism for TFEB governing pluripotency of mouse ESCs (mESCs) by regulating the pluripotency transcriptional network (PTN) in these cells. We observed high levels of TFEB mRNA and protein levels in undifferentiated mESCs. Interestingly, we found a reduction of Nanog and Sox2 levels in TFEB knockout (KO) mESCs while pluripotency was maintained as there was an upregulation of TFE3, a potent stem cell maintenance factor. In consistent, double knockout of TFEB/TFE3 (TFEB/3 DKO) reduced mESC pluripotency, as indicated by the loss of ESC morphology, reduction of ESC markers, and the emergence of differentiation markers. We further discovered that Nanog was a TFEB target gene in undifferentiated mESCs. TFEB also promoted sex-determining region Y-box2 (Sox2) transcription by forming a heterodimer with Sox2 in mESCs. Notably, Sox2, Oct4, and Nanog were also binding to the TFEB promoter and thus generating a feed-forward loop in relation to TFEB. Although high levels of nuclear TFEB are expected to enhance autophagy-lysosomal activity, undifferentiated mESC remarkably displayed low basal autophagy-lysosomal activity. Overexpression or knockout of TFEB did not affect the expression of TFEB lysosomal-autophagy target genes and TFEB also had a lesser binding affinity to its own lysosomal promoter-target genes in mESCs compared to differentiated cells. Collectively, these findings define a newly incorporative, moonlighting function for TFEB in regulating PTN, independent of its autophagy-lysosomal biogenesis roles.
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Project description:An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.
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Project description:The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.
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Project description:Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.
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Project description:BackgroundHistone cell cycle regulator (HIRA) complex is an important histone chaperone that mediates the deposition of the H3.3 histone variant onto chromatin independently from DNA synthesis. However, it is still unknown whether it participates in the expression control of retrotransposons and cell fate determination.MethodsWe screened the role of HIRA complex members in repressing the expression of retrotransposons by shRNA depletion in embryonic stem cells (ESCs) followed by RT-qPCR. RNA-seq was used to study the expression profiles after depletion of individual HIRA member. RT-qPCR and western blot were used to determine overexpression of HIRA complex members. Chromatin immunoprecipitation (ChIP)-qPCR was used to find the binding of H3.3, HIRA members to chromatin. Co-immunoprecipitation was used to identify the interaction between Hira mutant and Ubn2. ChIP-qPCR was used to identify H3.3 deposition change and western blot of chromatin extract was used to validate the epigenetic change. Bioinformatics analysis was applied for the analysis of available ChIP-seq data.ResultsWe revealed that Hira, Ubn2, and Ubn1 were the main repressors of 2-cell marker retrotransposon MERVL among HIRA complex members. Surprisingly, Ubn2 and Hira targeted different groups of retrotransposons and retrotransposon-derived long noncoding RNAs (lncRNAs), despite that they partially shared target genes. Furthermore, Ubn2 prevented ESCs to gain a 2-cell like state or activate trophectodermal genes upon differentiation. Mechanistically, Ubn2 and Hira suppressed retrotransposons by regulating the deposition of histone H3.3. Decreased H3.3 deposition, that was associated with the loss of Ubn2 or Hira, caused the reduction of H3K9me2 and H3K9me3, which are known repressive marks of retrotransposons.ConclusionsOverall, our findings shed light on the distinct roles of HIRA complex members in controlling retrotransposons and cell fate conversion in ESCs.
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Project description:The Retinoid inducible nuclear factor (Rinf), also known as CXXC5, is a nuclear protein, but its functions in the context of the chromatin are poorly defined. We find that in mouse embryonic stem cells (mESCs), Rinf binds to the chromatin and is enriched at promoters and enhancers of Tet1, Tet2, and pluripotency genes. The Rinf-bound regions show significant overlapping occupancy of pluripotency factors Nanog, Oct4, and Sox2, as well as Tet1 and Tet2. We found that Rinf forms a complex with Nanog, Oct4, Tet1, and Tet2 and facilitates their proper recruitment to regulatory regions of pluripotency and Tet genes in ESCs to positively regulate their transcription. Rinf deficiency in ESCs reduces expression of Rinf target genes, including several pluripotency factors and Tet enzymes, and causes aberrant differentiation. Together, our findings establish Rinf as a regulator of the pluripotency network genes and Tet enzymes in ESCs.
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Project description:Developmental gradients of morphogens and the formation of boundaries guide the choices between self-renewal and differentiation in stem cells. Still, surprisingly little is known about gene expression signatures of differentiating stem cells at the boundaries between regions. We thus combined inducible gene expression with a microfluidic technology to pattern gene expression in murine embryonic stem cells. Regional depletion of the Nanog transcriptional regulator was achieved through the exposure of cells to microfluidic gradients of morphogens. In this way, we established pluripotency-differentiation boundaries between Nanog expressing cells (pluripotency zone) and Nanog suppressed cells (early differentiation zone) within the same cell population, with a gradient of Nanog expression across the individual cell colonies, to serve as a mimic of the developmental process. Using this system, we identified strong interactions between Nanog and its target genes by constructing a network with Nanog as the root and the measured levels of gene expression in each region. Gene expression patterns at the pluripotency-differentiation boundaries recreated in vitro were similar to those in the developing blastocyst. This approach to the study of cellular commitment at the boundaries between gene expression domains, a phenomenon critical for understanding of early development, has potential to benefit fundamental research of stem cells and their application in regenerative medicine.
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Project description:Embryonic stem (ES) cells are pluripotent cells characterized by self-renewability and differentiation potential. Induced pluripotent stem (iPS) cells are ES cell-equivalent cells derived from somatic cells by the introduction of core reprogramming factors. ES and iPS cells are important sources for understanding basic biology and for generating therapeutic cells for clinical applications. Tribbles homolog 2 (Trib2) functions as a scaffold in signaling pathways. However, the relevance of Trib2 to the pluripotency of ES and iPS cells is unknown. In the present study, we elucidated the importance of Trib2 in maintaining pluripotency in mouse ES cells and in generating iPS cells from somatic cells through the reprogramming process. Trib2 expression decreased as ES cells differentiated, and Trib2 knockdown in ES cells changed their colony morphology while reducing the activity of alkaline phosphatase and the expression of the pluripotency marker genes Oct4, Sox2, Nanog and Klf4. Trib2 directly interacted with Oct4 and elevated Oct4 promoter activity. During the generation of iPS cells, Trib2 knockdown decreased the reprogramming efficiency of mouse embryonic fibroblasts, whereas Trib2 overexpression significantly increased their reprogramming efficiency. In summary, our results suggest that Trib2 is important for maintaining self-renewal in ES cells and for pluripotency induction during the reprogramming process.
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Project description:Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency-naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten-/- mouse ESCs with the CRISPR-Cas9 system and verified that Pten-/- ESCs maintained naïve pluripotency by blocking Gsk3? activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3? modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.
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Project description:Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1beta (TIF1beta), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4-dependent transcription in a phosphorylation-dependent manner. TIF1beta is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1beta is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcd1 [corrected], Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1beta specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1beta regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1beta provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.
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