Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in ...differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.
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•Human iPSCs erase aging signatures and hiPSC-derived neurons remain rejuvenated•Directly converted iNs preserve donor age-dependent transcriptomic signatures•Nuclear transport receptor RanBP17 is decreased in aged human cells and iNs•Aged and RanBP17-depleted cells show nucleocytoplasmic compartmentalization defects
Mertens and colleagues compare transcriptomes of human fibroblasts, induced neurons (iNs), iPSCs, iPSC-derived neurons, and brain samples from a broad range of aged donors, finding that iNs retain donor aging signatures, while iPSCs are rejuvenated. RanBP17 was consistently decreased during aging, leading to compromised nucleocytoplasmic compartmentalization in aged human cells.
Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of ...the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including ...the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.
The Polycomb-group (PcG) repressive complex-1 (PRC1) forms microscopically visible clusters in nuclei; however, the impact of this cluster formation on transcriptional regulation and the underlying ...mechanisms that regulate this process remain obscure. Here, we report that the sterile alpha motif (SAM) domain of a PRC1 core component Phc2 plays an essential role for PRC1 clustering through head-to-tail macromolecular polymerization, which is associated with stable target binding of PRC1/PRC2 and robust gene silencing activity. We propose a role for SAM domain polymerization in this repression by two distinct mechanisms: first, through capturing and/or retaining PRC1 at the PcG targets, and second, by strengthening the interactions between PRC1 and PRC2 to stabilize transcriptional repression. Our findings reveal a regulatory mechanism mediated by SAM domain polymerization for PcG-mediated repression of developmental loci that enables a robust yet reversible gene repression program during development.
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•PRC1 forms visible subnuclear clusters at its target loci in mouse primary cells•The polymerization of the Phc2 SAM domain is required for PRC1 clustering•Clustering of PRC1 links to chromatin condensation and gene silencing•PRC1 clustering associates with stable binding of PRC1/PRC2 at its target loci
Gene silencing by the Polycomb-repressive complex-1 (PRC1) is crucial for embryogenesis. Isono et al. show that subnuclear PRC1 clustering at its target genes is mediated by the polymerization capacity of the Phc2 SAM domain and associates with stable PRC1/PRC2 binding, trimethylation of histone H3 Lys27, and robust gene silencing.
Single-cell transcriptomics of neocortical neurons have revealed more than 100 clusters corresponding to putative cell types. For inhibitory and subcortical projection neurons (SCPNs), there is a ...strong concordance between clusters and anatomical descriptions of cell types. In contrast, cortico-cortical projection neurons (CCPNs) separate into surprisingly few transcriptomic clusters, despite their diverse anatomical projection types. We used projection-dependent single-cell transcriptomic analyses and monosynaptic rabies tracing to compare mouse primary visual cortex CCPNs projecting to different higher visual areas. We find that layer 2/3 CCPNs with different anatomical projections differ systematically in their gene expressions, despite forming only a single genetic cluster. Furthermore, these neurons receive feedback selectively from the same areas to which they project. These findings demonstrate that gene-expression analysis in isolation is insufficient to identify neuron types and have important implications for understanding the functional role of cortical feedback circuits.
•V1 CCPNs display differential gene expression depending on their projections•V1 CCPNs receive feedback inputs from the same areas to which they project•Gene-expression analysis in isolation is insufficient to identify neuron types
Kim et al. show that neuronal cell groups from within a single transcriptomic cluster can be further separated based on their anatomical projections and connectivity patterns. Connectivity is an important feature for cell-type identification. Gene expression alone cannot be used to fully annotate cortical cell types.
Somatic cells can be reprogrammed to a pluripotent state through the ectopic expression of defined transcription factors. Understanding the mechanism and kinetics of this transformation may shed ...light on the nature of developmental potency and suggest strategies with improved efficiency or safety. Here we report an integrative genomic analysis of reprogramming of mouse fibroblasts and B lymphocytes. Lineage-committed cells show a complex response to the ectopic expression involving induction of genes downstream of individual reprogramming factors. Fully reprogrammed cells show gene expression and epigenetic states that are highly similar to embryonic stem cells. In contrast, stable partially reprogrammed cell lines show reactivation of a distinctive subset of stem-cell-related genes, incomplete repression of lineage-specifying transcription factors, and DNA hypermethylation at pluripotency-related loci. These observations suggest that some cells may become trapped in partially reprogrammed states owing to incomplete repression of transcription factors, and that DNA de-methylation is an inefficient step in the transition to pluripotency. We demonstrate that RNA inhibition of transcription factors can facilitate reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the overall efficiency of the reprogramming process.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Direct conversion of human somatic fibroblasts into induced neurons (iNs) allows for the generation of functional neurons while bypassing any stem cell intermediary stages. Although iN technology has ...an enormous potential for modeling age-related diseases, as well as therapeutic approaches, the technology faces limitations due to variable conversion efficiencies and a lack of thorough understanding of the signaling pathways directing iN conversion. Here, we introduce a new all-in-one inducible lentiviral system that simplifies fibroblast transgenesis for the two pioneer transcription factors, Ngn2 and Ascl1, and markedly improves iN yields. Further, our timeline RNA-Seq data across the course of conversion has identified signaling pathways that become transcriptionally enriched during iN conversion. Small molecular modulators were identified for four signaling pathways that reliably increase the yield of iNs. Taken together, these advances provide an improved toolkit for iN technology and new insight into the mechanisms influencing direct iN conversion.
Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited ...to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain ...insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.
► Ectopic Oct4, Sox2, Klf4, and c-Myc induce H3K4 methylation at developmental promoters ► Promoter H3K4 methylation precedes transcriptional activation ► Localized depletion of H3K27me3 occurs within CpG island promoters that gain H3K4me2 ► H3K4me2 enhancer signatures are gained and lost in response to OSKM factor induction
Despite widespread interest in using human induced pluripotent stem cells (hiPSCs) in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we ...report a comprehensive and efficient differentiation paradigm for hiPSCs that generate multiple CA3 pyramidal neuron subtypes as detected by single-cell RNA sequencing (RNA-seq). This differentiation paradigm exhibits characteristics of neuronal network maturation, and rabies virus tracing revealed synaptic connections between stem cell-derived dentate gyrus (DG) and CA3 neurons in vitro recapitulating the neuronal connectivity within the hippocampus. Because hippocampal dysfunction has been implicated in schizophrenia, we applied DG and CA3 differentiation paradigms to schizophrenia-patient-derived hiPSCs. We detected reduced activity in DG-CA3 co-culture and deficits in spontaneous and evoked activity in CA3 neurons from schizophrenia-patient-derived hiPSCs. Our approach offers critical insights into the network activity aspects of schizophrenia and may serve as a promising tool for modeling diseases with hippocampal vulnerability.
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•Differentiation of human CA3 pyramidal neuron is modeled using ESCs/iPSCs•RNA-seq and immunocytochemistry revealed pyramidal neuron diversity•Rabies virus tracing showed a connection between ESC-derived DG and CA3 neurons•Schizophrenia hiPSC-derived DG-CA3 co-cultures present deficits in hippocampal activity
Sarkar et al. established a differentiation paradigm that generates human CA3 pyramidal neurons from ESCs and iPSCs and recapitulates hippocampal connectivity in vitro. This work reveals reduced levels of activity of schizophrenia-patient-derived neurons, offering opportunities for modeling diseases with hippocampal vulnerability.
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