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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (scRNA-Seq)

ABSTRACT: Many lincRNAs have been identified as essential regulators for ESC pluripotency through siRNA-based screening. However, the biological and molecular functions of the large intergenic noncoding RNAs (lincRNAs) remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 previously annotated lincRNAs in ESCs. Unexpectedly, it was found that most lincRNAs are dispensable for pluripotency maintenance, cloning formation ability, proliferation and apoptosis in ESCs when we disrupted these lincRNAs individually or in combinations. Single-cell transcriptomic analysis of a set of lncRNA knockout ESCs showed consistent results, indicating that previously observed functions of lincRNAs on ESC pluripotency maintenance may be due to off-targeted effects. Interestingly, deletion and transcriptional repression of linc1343 disrupts embryoid body (EB) differentiation in ESC. Notably, GM17300 and two small RNAs (Snora73a, Snora73b) derived from the lnc1343 locus are highly expressed in ESCs. Further overexpression of Snora73a and Snora73b in linc1343 knockout cells rescue the phenotype, suggesting that snoRNAs derived from linc1343 are the responders for EB differentiation. Taken together, our results demonstrate the divergent effects of lincRNAs on regulating ESC pluripotency and EB differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE225238 | GEO | 2024/08/07

REPOSITORIES: GEO

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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (RNA-Seq)

Project description:Many lincRNAs have been identified as essential regulators for ESC pluripotency through siRNA-based screening. However, the biological and molecular functions of the large intergenic noncoding RNAs (lincRNAs) remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 previously annotated lincRNAs in ESCs. Unexpectedly, it was found that most lincRNAs are dispensable for pluripotency maintenance, cloning formation ability, proliferation and apoptosis in ESCs when we disrupted these lincRNAs individually or in combinations. Single-cell transcriptomic analysis of a set of lncRNA knockout ESCs showed consistent results, indicating that previously observed functions of lincRNAs on ESC pluripotency maintenance may be due to off-targeted effects. Interestingly, deletion and transcriptional repression of linc1343 disrupts embryoid body (EB) differentiation in ESC. Notably, GM17300 and two small RNAs (Snora73a, Snora73b) derived from the lnc1343 locus are highly expressed in ESCs. Further overexpression of Snora73a and Snora73b in linc1343 knockout cells rescue the phenotype, suggesting that snoRNAs derived from linc1343 are the responders for EB differentiation. Taken together, our results demonstrate the divergent effects of lincRNAs on regulating ESC pluripotency and EB differentiation.

2024-08-07 | GSE225237 | GEO

lincRNAs act in the circuitry controlling pluripotency and differentiation

Project description:While thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem cells (ESC) and characterized the effects on gene expression. Here we show that knockdown of lincRNAs have major consequences on gene expression patterns, comparable to knockdown of well-known ESC regulators. Notably, lincRNAs primarily affect gene expression in trans. We identify dozens of lincRNAs whose knockdown causes an exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ESCs and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts physically bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ESC state. We generated five lentiviral-based shRNAs targeting each of the 237 lincRNAs previously identified in ESCs. These shRNAs successfully targeted 147 lincRNAs and reduced their expression by an average of ~75% compared to endogenous levels in ESCs. As positive controls, we generated shRNAs targeting ~50 genes encoding regulatory proteins, including both transcription factor and chromatin factor genes that have been shown to play critical roles in ESC regulation; we obtained validated hairpins against 40 of these genes. As negative controls, we performed independent infections with lentiviruses containing 27 different shRNAs with no known cellular target RNA.

2011-08-29 | E-GEOD-30245 | biostudies-arrayexpress

lincRNAs act in the circuitry controlling pluripotency and differentiation

2011-08-30 | GSE30245 | GEO

A mass spectrometry survey of chromatin-associated proteins in pluripotency and early lineage commitment

Project description:Pluripotent Embryonic Stem Cells (ESCs) can be captured in vitro in different states, ranging from unrestricted ‘naïve’ to more developmentally constrained ‘primed’ pluripotency. Complexes involved in epigenetic regulation and key transcription factors have been shown to be involved in specifying these distinct states. In this study, we use proteomic profiling of the chromatin landscape in naive pluripotent ESCs, Epistem cells (EpiSCs) and early differentiated ESCs to survey the chromatin in naïve and primed pluripotency and during differentiation. We provide a comprehensive overview of epigenetic complexes situated on the chromatin and identify proteins associated with the maintenance and loss of pluripotency. The findings presented here indicate major compositional alterations of epigenetic complexes starting from ESC priming onwards. Our results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.

2019-07-04 | PXD011782 | Pride

Zfp462 regulates the pluripotency of embryonic stem cells by cooperating with the core transcriptional network

Project description:Zfp462 is a vertebrate-specific C2H2-type transcription factor that has been found to play a crucial role in chromatin assembly and heterochromatin-mediated transcriptional silencing. However, its exact function in regulating the pluripotency of embryonic stem cells (ESCs) remains poorly understood. In this study, we observe that Zfp462 is highly expressed in undifferentiated ESCs, gradually decreases upon pluripotency exit, but then increases again during lineage differentiation. Knocking out of Zfp462 leads to reduced self-renewal of ESCs, abnormal pluripotency exit, and imbalance of lineage specification/differentiation. Multi-omics integrative analysis suggests that Zfp462 may function as a component of the ESC CORE module and collaborate with Oct4/Sox2/Nanog (OSN) to regulate the expression of pluripotency and developmental genes. Collectively, our research not only reveals the functions of Zfp462 in ESC self-renewal and differentiation but also proposes Zfp462 as a novel player in the pluripotency gene regulatory network (PGRN).

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Polycomb protein EED is required for selective silencing of pluripotency genes upon ESC differentiation

Project description:Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation. RNAs obtained from undifferentiated (d0) wild type and Eed KO ESCs and from day 3 (d3) and day 7 (d7) respective Ebs were subjected to Affymetrix Mouse Gene 1.0 ST Array. 24 samples in total.

2014-08-28 | E-GEOD-49305 | biostudies-arrayexpress

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Project description:Embryonic stem cells (ESC) are able to give rise to any somatic cell type. A lot is known about how ESC pluripotency is maintained, but comparatively less is known about how differentiation is promoted. Cell fate decisions are regulated by interactions between signalling and transcriptional networks. Recent studies have shown that the overexpression or downregulation of the transcription factor Jun can affect the ESC fate. Here we have focussed on the role of the Jun in the exit of mouse ESCs from ground state pluripotency and the onset of early differentiation. Transcriptomic analysis of differentiating ESCs reveals that Jun is required to upregulate a programme of genes associated with cell adhesion as ESCs exit the pluripotent ground state.

2015-12-07 | E-MTAB-4068 | biostudies-arrayexpress

Polycomb protein EED is required for selective silencing of pluripotency genes upon ESC differentiation

2014-08-28 | GSE49305 | GEO

Pluripotency-associated miR-290/302 family of microRNAs promote the dismantling of naive pluripotency

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2018-11-29 | GSE123096 | GEO

Expression data from control (F/F) and Snai1 knockout (KO) embryoid body (EB) and teratoma ™

Project description:Snai1 is a master factor of epithelial to mesenchymal transitioin (EMT), however, its role in embryonic stem cell (ESC) differentiation and lineage commitment remains undefined. We used microarrays to compare the global programme of gene expression between control and Snai1 knockout ESCs-derived EB and teratoma. For EBs, control and Snai1 knockout ESCs were cultured as embryoid bodies in spotaneous differentiation media, RNA of 5 days EBs were collected for Affymetrix microarrays. For teratomas, control and Snai1 knockout ESCs were injected into nude mice to form teratomas. RNA of 6 weeks were collected for Affymetrix microarrays.

2013-12-10 | E-GEOD-53106 | biostudies-arrayexpress

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