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Stem Cell Epigenetics News

September 2016


MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. The authors of this recent Nature Scientific Reports publication have developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. As such, a new RNA tool, miR-switch, was synthesized as a live-cell reporter mRNA for miR-302a activity. This new tool can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and prevent teratoma formation in an in vivo tumorigenicity assay. This system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells and holds great potential for investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.
Parr et al. (2016) Nat Scientific Reports. DOI: 10.1038/srep32532
Abstract.
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Human Disease Modeling Reveals Integrated Transcriptional and Epigenetic Mechanisms of NOTCH1 Haploinsufficiency
The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. In this recent Cell publication, human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) were utilized to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Computational predictions of the disrupted NOTCH1-dependent gene network reveal regulatory nodes that, when modulated, restored the network toward the NOTCH1+/+ state. The results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.
Theodoris et al. (2016) , Cell. http://dx.doi.org/10.1016/j.cell.2015.02.035
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Cellular Metabolism and Induced Pluripotency
Cellular metabolism involves a set of complex and highly coordinated life-sustaining biochemical reactions that convert or use energy to maintain the living state of cells. Cellular metabolism has long been considered as a consequence, rather than a driver, of cell-fate changes—a view that has recently been challenged by its intrinsic links to epigenetic modifications of chromatin during development, disease progression, and cellular reprograming. In this Review, the authors summarize what we know about metabolic pathways characteristic of pluripotent stem cells (PSCs) and discuss metabolic reprograming to induced pluripotency and the modeling of metabolic diseases with iPSCs.
Wu et al. (2016) Cell. http://dx.doi.org/10.1016/j.cell.2016.08.008
Abstract.
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August 2016


Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment
Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. A group of scientists from Cologne, Germany show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. These results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.
Le et al. (2016) Nat Cell Biol. doi:10.1038/ncb3387.
Abstract.
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DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells
Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation-hydroxymethylation pathway. Using Dnmt3a and Tet2 double-knockout mice in which the development of malignancy is accelerated, researchers at the Baylor College of Medicine in Houston, Texas show that the double-knockout methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulators Klf1 and Epor, is upregulated in double-knockout hematopoietic stem cells (HSCs). DNMT3A and TET2 both repress Klf1, suggesting a model of cooperative inhibition by epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, the loss of which, along with DNMT3A, obstructs differentiation, leading to transformation.
Zhang et al. (2016) , Nat Genet. doi:10.1038/ng.3610.
Abstract.
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5-Hydroxymethylcytosine remodeling precedes lineage specification during differentiation of human CD4(+) T cells
5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, scientists from Linking University in Sweden report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. They observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Their results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment.
Nestor et al. (2016) Cell Rep. doi:10.1016/j.celrep.2016.05.091.
Abstract.
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July 2016


Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene-expression program and acute leukemia development
DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, researchers report that the DNMT3A mutational hotspot at Arg882 (DNMT3AR882H) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3AR882H directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3AR882H induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3AR882H-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3AR882H-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.
Lu et al. (2016) Cancer Cell. doi:10.1016/j.ccell.2016.05.008.
Abstract.
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Zfp281 coordinates opposing functions of Tet1 and Tet2 in pluripotent states
Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, the authors performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. They identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. The study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states.
Fidalgo et al. (2016) , Cell Stem Cell. doi:10.1016/j.stem.2016.01.007.
Abstract.
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Regulation of the DNA methylation landscape in human somatic cell reprogramming by the miR-29 family
Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, scientist found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Their global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. These findings uncover an important miRNA-based approach to generate clinically robust iPSCs.
Hysolli et al. (2016) Stem Cell Reports. doi:10.1016/j.stemcr.2016.05.014.
Abstract.
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June 2016


Local genome topology can exhibit an incompletely rewired 3D-folding state during somatic cell reprogramming
Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here researchers from the laboratory of Dr. Phillips-Cremins at the University of Pennsylvania integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). They find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in their iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, the authors identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. These results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.
Beagan et al. (2016) Cell Stem Cell. doi:10.1016/j.stem.2016.04.004.
Abstract.
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Cell-of-origin-specific 3D genome structure acquired during somatic cell reprogramming
Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here researchers studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. They find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as they also find for cell-of-origin-dependent gene expression patterns.
Krijger et al. (2016) , Cell Stem Cell. doi:10.1016/j.stem.2016.01.007.
Abstract.
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Epigenetic reprogramming of fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution
The exact timing and contribution of epigenetic reprogramming to carcinogenesis are unclear. Women harbouring BRCA1/2 mutations demonstrate a 30-40-fold increased risk of high-grade serous extra-uterine Müllerian cancers (HGSEMC), otherwise referred to as 'ovarian carcinomas', which frequently develop from fimbrial cells but not from the proximal portion of the fallopian tube. Here the authors compare the DNA methylome of the fimbrial and proximal ends of the fallopian tube in BRCA1/2 mutation carriers and non-carriers. They show that the number of CpGs displaying significant differences in methylation levels between fimbrial and proximal fallopian tube segments are threefold higher in BRCA mutation carriers than in controls, correlating with overexpression of activation-induced deaminase in their fimbrial epithelium. The differentially methylated CpGs accurately discriminate HGSEMCs from non-serous subtypes. Epigenetic reprogramming is an early pre-malignant event integral to BRCA1/2 mutation-driven carcinogenesis. Their findings may provide a basis for cancer-preventative strategies.
Bartlett et al. (2016) Nat Commun. doi:10.1038/ncomms11620.
Abstract.
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May 2016


Suppression of enhancer overactivation by a RACK7-histone demethylase complex
Regulation of enhancer activity is important for controlling gene expression programs. Here, results from a collaboration between scientists at Harvard University and Fudan University in Shanghai report that a biochemical complex containing a potential chromatin reader, RACK7, and the histone lysine 4 tri-methyl (H3K4me3)-specific demethylase KDM5C occupies many active enhancers, including almost all super-enhancers. Loss of RACK7 or KDM5C results in overactivation of enhancers, characterized by the deposition of H3K4me3 and H3K27Ac, together with increased transcription of eRNAs and nearby genes. Furthermore, loss of RACK7 or KDM5C leads to derepression of S100A oncogenes and various cancer-related phenotypes. Their findings reveal a RACK7/KDM5C-regulated, dynamic interchange between histone H3K4me1 and H3K4me3 at active enhancers, representing an additional layer of regulation of enhancer activity. The authors propose that RACK7/KDM5C functions as an enhancer “brake” to ensure appropriate enhancer activity, which, when compromised, could contribute to tumorigenesis.
Shen et al. (2016) Cell. doi:10.1016/j.cell.2016.02.064.
Abstract.
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The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation
Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. Researchers from the University of Pennsylvania combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. They find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.
Iwafuchi-Doi et al. (2016) Mol Cell. doi:10.1016/j.molcel.2016.03.001.
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An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, the authors investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Their data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, this study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.
Petell et al. (2016) Nucleic Acids Res. doi:10.1093/nar/gkw426.
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