ABSTRACT
Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.
Subject(s)
Cognition , Embryo, Mammalian , Embryonic Development , Histone Demethylases , Wnt Signaling Pathway , Animals , Humans , Mice , Anxiety , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Embryo, Mammalian/metabolism , Gene Expression Profiling , Histone Demethylases/genetics , Histone Demethylases/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intellectual Disability/genetics , Memory , Mice, Knockout , Mutation , Neurogenesis/genetics , Wnt Signaling Pathway/drug effectsABSTRACT
Gene regulatory networks are pivotal for many biological processes. In mouse embryonic stem cells (mESCs), the transcriptional network can be divided into three functionally distinct modules: Polycomb, Core, and Myc. The Polycomb module represses developmental genes, while the Myc module is associated with proliferative functions, and its mis-regulation is linked to cancer development. Here, we show that, in mESCs, the Polycomb repressive complex 2 (PRC2)-associated protein EPOP (Elongin BC and Polycomb Repressive Complex 2-associated protein; a.k.a. C17orf96, esPRC2p48, and E130012A19Rik) co-localizes at chromatin with members of the Myc and Polycomb module. EPOP interacts with the transcription elongation factor Elongin BC and the H2B deubiquitinase USP7 to modulate transcriptional processes in mESCs similar to MYC. EPOP is commonly upregulated in human cancer, and its loss impairs the proliferation of several human cancer cell lines. Our findings establish EPOP as a transcriptional modulator, which impacts both Polycomb and active gene transcription in mammalian cells.
Subject(s)
Chromatin/chemistry , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/genetics , Polycomb Repressive Complex 2/genetics , Transcription Factors/genetics , Ubiquitin-Specific Proteases/genetics , Animals , Cell Differentiation , Cell Line, Tumor , Chromatin/metabolism , Chromosomal Proteins, Non-Histone , DNA Polymerase II/genetics , DNA Polymerase II/metabolism , Elongin , Embryo, Mammalian , Gene Regulatory Networks , Histones/genetics , Histones/metabolism , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nerve Tissue Proteins/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic , Protein Binding , Transcription Factors/metabolism , Transcription, Genetic , Ubiquitin-Specific Peptidase 7 , Ubiquitin-Specific Proteases/metabolismABSTRACT
NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.
Subject(s)
Chromatin/enzymology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Mouse Embryonic Stem Cells/metabolism , Neurogenesis/genetics , Proto-Oncogene Proteins/metabolism , RNA-Binding Proteins/physiology , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Animals , Cells, Cultured , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Expression Regulation , Gene Knockout Techniques , Mice , Proto-Oncogene Proteins/chemistry , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Seq , Transcription, GeneticABSTRACT
NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network.
Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/metabolism , Gene Expression Regulation/physiology , Gene Regulatory Networks/genetics , Homeodomain Proteins/metabolism , Pluripotent Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation/genetics , Chromatin Immunoprecipitation , Feedback, Physiological , Flow Cytometry , Gene Expression Regulation/genetics , Green Fluorescent Proteins , In Situ Hybridization, Fluorescence , Mice , Nanog Homeobox Protein , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency.
Subject(s)
DNA-Binding Proteins/metabolism , MAP Kinase Signaling System , Mouse Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Self Renewal , Enzyme Activation , Epigenesis, Genetic , Gene Expression Profiling , Genome , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Nanog Homeobox Protein/metabolism , Phosphorylation , RNA-Binding Proteins , Transcriptome/geneticsABSTRACT
Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4(+/-) ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation.
Subject(s)
Enhancer Elements, Genetic/genetics , Homeodomain Proteins/metabolism , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Signal Transduction , Base Sequence , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Clone Cells , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Humans , Molecular Sequence Data , Pluripotent Stem Cells/drug effects , Protein Binding/drug effects , Serum , Signal Transduction/drug effects , Wnt Proteins/metabolismABSTRACT
Embryonic stem cell (ESC) self-renewal efficiency is determined by the level of Nanog expression. However, the mechanisms by which Nanog functions remain unclear, and in particular, direct Nanog target genes are uncharacterized. Here we investigate ESCs expressing different Nanog levels and Nanog(-/-) cells with distinct functionally inducible Nanog proteins to identify Nanog-responsive genes. Surprisingly, these constitute a minor fraction of genes that Nanog binds. Prominent among Nanog-reponsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding of RNAPolII, and stimulates Esrrb transcription. Overexpression of Esrrb in ESCs maintains cytokine-independent self-renewal and pluripotency. Remarkably, this activity is retained in Nanog(-/-) ESCs. Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal. These findings are consistent with the functional placement of Esrrb downstream of Nanog.
Subject(s)
Homeodomain Proteins/metabolism , Neural Stem Cells/physiology , Pluripotent Stem Cells/physiology , Receptors, Estrogen/metabolism , Animals , Cell Fusion , Cell Line , Cell Proliferation , Cell Survival/genetics , Cellular Reprogramming/genetics , Chimera , Embryo Culture Techniques , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Interleukin-6/metabolism , Mice , Microarray Analysis , Mutant Proteins/genetics , Nanog Homeobox Protein , Receptors, Estrogen/genetics , Receptors, OSM-LIF/genetics , Transgenes/geneticsABSTRACT
During mouse embryogenesis, reversion of imprinted X chromosome inactivation in the pluripotent inner cell mass of the female blastocyst is initiated by the repression of Xist from the paternal X chromosome. Here we report that key factors supporting pluripotency-Nanog, Oct3/4, and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem (ES) cells. Whereas Nanog null ES cells display a reversible and moderate up-regulation of Xist in the absence of any apparent modification of Oct3/4 and Sox2 binding, the drastic release of all three factors from Xist intron 1 triggers rapid ectopic accumulation of Xist RNA. We conclude that the three main genetic factors underlying pluripotency cooperate to repress Xist and thus couple X inactivation reprogramming to the control of pluripotency during embryogenesis.