We developed an affinity purification approach to isolate tagged nuclei in mice (similar to INTACT; [Deal R.B. and Henikoff S. A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell 18,1030-1040. (2010)]) and used it to characterize genome-wide patterns of transcription, DNA methylation, and chromatin accessibility in 3 major neuron classes of the neocortex (excitatory pyramidal neurons, parvalbumin (PV)-positive GABAergic interneurons, and vasoactive intestinal peptide (VIP)-positive GABAergic interneurons). By combining cell purification and integrative analysis, our findings relate the phenotypic and functional complexity of neocortical neurons to their underlying transcriptional and epigenetic diversity. Overall design: RNA-seq, MethylC-seq, ATAC-seq, and ChIP-seq for histone modifications using INTACT-purified nuclei from the mouse neocortex
Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain.
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Epigenetic polymorphism and the stochastic formation of differentially methylated regions in normal and cancerous tissues.
Specimen part, Cell line
View SamplesDNA methylation has been comprehensively profiled in normal and cancer cells, but the dynamics that form, maintain and reprogram differentially methylated regions remain enigmatic. We show that methylation patterns within populations of cells from individual somatic tissues are heterogeneous and polymorphic. Using in vitro evolution of immortalized fibroblasts for over 300 generations, we track the dynamics of polymorphic methylation at regions developing significant differential methylation on average. The data indicate that changes in population-averaged methylation occur through a stochastic process that generates a stream of local and uncorrelated methylation aberrations. Despite the stochastic nature of the process, nearly deterministic epigenetic remodeling emerges on average at loci that lose or gain resistance to methylation accumulation. Changes in the susceptibility to methylation accumulation are correlated with changes in histone modifications and CTCF occupancy. Characterizing epigenomic polymorphism within cell populations is therefore critical for understanding methylation dynamics in normal and cancer cells.
Epigenetic polymorphism and the stochastic formation of differentially methylated regions in normal and cancerous tissues.
Specimen part, Cell line
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Corrigendum: Deterministic direct reprogramming of somatic cells to pluripotency.
Specimen part
View SamplesSomatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution.
Deterministic direct reprogramming of somatic cells to pluripotency.
Specimen part
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Derivation of novel human ground state naive pluripotent stem cells.
Specimen part, Cell line
View SamplesMouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3b signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters.Upon withdrawal of 2i/LIF, nave mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalency acquisition on lineage regulatory genes. The feasibility for establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in rodent ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Derivation of novel human ground state naive pluripotent stem cells.
Specimen part
View SamplesAs a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array.
Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma.
Cell line, Treatment
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CFC1 is a cancer stemness-regulating factor in neuroblastoma.
Specimen part, Cell line, Subject
View SamplesLoss or reduction in function of tumor suppressor genes contributes to tumorigenesis. We identified a novel homozygous deletion of DACH1 in gliomas.
Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells.
Specimen part, Cell line
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