Stress induces behavioral abnormalities by increasing expression of phagocytic receptor MERTK in astrocytes to promote synapse phagocytosis.

Childhood neglect and/or abuse can induce mental health conditions with unknown mechanisms. Here, we identified stress hormones as strong inducers of astrocyte-mediated synapse phagocytosis. Using in vitro, in vivo, and human brain organoid experiments, we showed that stress hormones increased the expression of the Mertk phagocytic receptor in astrocytes through glucocorticoid receptor (GR). In post-natal mice, exposure to early social deprivation (ESD) specifically activated the GR-MERTK pathway in astrocytes, but not in microglia. The excitatory post-synaptic density in cortical regions was reduced in ESD mice, and there was an increase in the astrocytic engulfment of these synapses. The loss of excitatory synapses, abnormal neuronal network activities, and behavioral abnormalities in ESD mice were largely prevented by ablating GR or MERTK in astrocytes. Our work reveals the critical roles of astrocytic GR-MERTK activation in evoking stress-induced abnormal behaviors in mice, suggesting GR-MERTK signaling as a therapeutic target for stress-induced mental health conditions.

Light-stimulated insulin secretion from pancreatic islet-like organoids derived from human pluripotent stem cells.

Optogenetic techniques permit non-invasive, spatiotemporal, and reversible modulation of cellular activities. Here, we report a novel optogenetic regulatory system for insulin secretion in human pluripotent stem cell (hPSC)-derived pancreatic islet-like organoids using monSTIM1 (monster-opto-Stromal interaction molecule 1), an ultra-light-sensitive OptoSTIM1 variant. The monSTIM1 transgene was incorporated at the AAVS1 locus in human embryonic stem cells (hESCs) by CRISPR-Cas9-mediated genome editing. Not only were we able to elicit light-induced intracellular Ca2+ concentration ([Ca2+]i) transients from the resulting homozygous monSTIM1+/+-hESCs, but we also successfully differentiated them into pancreatic islet-like organoids (PIOs). Upon light stimulation, the ß-cells in these monSTIM1+/+-PIOs displayed reversible and reproducible [Ca2+]i transient dynamics. Furthermore, in response to photoexcitation, they secreted human insulin. Light-responsive insulin secretion was similarly observed in monSTIM1+/+-PIOs produced from neonatal diabetes (ND) patient-derived induced pluripotent stem cells (iPSCs). Under LED illumination, monSTIM1+/+-PIO-transplanted diabetic mice produced human c-peptide. Collectively, we developed a cellular model for the optogenetic control of insulin secretion using hPSCs, with the potential to be applied to the amelioration of hyperglycemic disorders.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Association between ARID2 and RAS-MAPK pathway in intellectual disability and short stature.

BACKGROUND: ARID2 belongs to the Switch/sucrose non-fermenting complex, in which the genetic defects have been found in patients with dysmorphism, short stature and intellectual disability (ID). As the phenotypes of patients with ARID2 mutations partially overlap with those of RASopathy, this study evaluated the biochemical association between ARID2 and RAS-MAPK pathway. METHODS: The phenotypes of 22 patients with either an ARID2 heterozygous mutation or haploinsufficiency were reviewed. Comprehensive molecular analyses were performed using somatic and induced pluripotent stem cells (iPSCs) of a patient with ARID2 haploinsufficiency as well as using the mouse model of Arid2 haploinsufficiency by CRISPR/Cas9 gene editing. RESULTS: The phenotypic characteristics of ARID2 deficiency include RASopathy, Coffin-Lowy syndrome or Coffin-Siris syndrome or undefined syndromic ID. Transient ARID2 knockout HeLa cells using an shRNA increased ERK1 and ERK2 phosphorylation. Impaired neuronal differentiation with enhanced RAS-MAPK activity was observed in patient-iPSCs. In addition, Arid2 haploinsufficient mice exhibited reduced body size and learning/memory deficit. ARID2 haploinsufficiency was associated with reduced IFITM1 expression, which interacts with caveolin-1 (CAV-1) and inhibits ERK activation. DISCUSSION: ARID2 haploinsufficiency is associated with enhanced RAS-MAPK activity, leading to reduced IFITM1 and CAV-1 expression, thereby increasing ERK activity. This altered interaction might lead to abnormal neuronal development and a short stature.

Aberrant Cortical Layer Development of Brain Organoids Derived from Noonan Syndrome-iPSCs.

Noonan syndrome (NS) is a genetic disorder mainly caused by gain-of-function mutations in Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Although diverse neurological manifestations are commonly diagnosed in NS patients, the mechanisms as to how SHP2 mutations induce the neurodevelopmental defects associated with NS remain elusive. Here, we report that cortical organoids (NS-COs) derived from NS-induced pluripotent stem cells (iPSCs) exhibit developmental abnormalities, especially in excitatory neurons (ENs). Although NS-COs develop normally in their appearance, single-cell transcriptomic analysis revealed an increase in the EN population and overexpression of cortical layer markers in NS-COs. Surprisingly, the EN subpopulation co-expressing the upper layer marker SATB2 and the deep layer maker CTIP2 was enriched in NS-COs during cortical development. In parallel with the developmental disruptions, NS-COs also exhibited reduced synaptic connectivity. Collectively, our findings suggest that perturbed cortical layer identity and impeded neuronal connectivity contribute to the neurological manifestations of NS.

Exosome-mediated activation of toll-like receptor 3 in stellate cells stimulates interleukin-17 production by γδ T cells in liver fibrosis.

UNLABELLED: During liver injury, hepatocytes secrete exosomes that include diverse types of self-RNAs. Recently, self-noncoding RNA has been recognized as an activator of Toll-like receptor 3 (TLR3). However, the roles of hepatic exosomes and TLR3 in liver fibrosis are not yet fully understood. Following acute liver injury and early-stage liver fibrosis induced by a single or 2-week injection of carbon tetrachloride (CCl4 ), increased interleukin (IL)-17A production was detected primarily in hepatic γδ T cells in wild-type (WT) mice. However, liver fibrosis and IL-17A production by γδ T cells were both significantly attenuated in TLR3 knockout (KO) mice compared with WT mice. More interestingly, IL-17A-producing γδ T cells were in close contact with activated hepatic stellate cells (HSCs), suggesting a role for HSCs in IL-17A production by γδ T cells. In vitro treatments with exosomes derived from CCl4 -treated hepatocytes significantly increased the expression of IL-17A, IL-1ß, and IL-23 in WT HSCs but not in TLR3 KO HSCs. Furthermore, IL-17A production by γδ T cells was substantially increased upon coculturing with exosome-treated WT HSCs or conditioned medium from TLR3-activated WT HSCs. However, similar increases were not detected when γδ T cells were cocultured with exosome-treated HSCs from IL-17A KO or TLR3 KO mice. Using reciprocal bone marrow transplantation between WT and TLR3 KO mice, we found that TLR3 deficiency in HSCs contributed to decreased IL-17A production by γδ T cells, as well as liver fibrosis. CONCLUSION: In liver injury, the exosome-mediated activation of TLR3 in HSCs exacerbates liver fibrosis by enhancing IL-17A production by γδ T cells, which might be associated with HSC stimulation by unknown self-TLR3 ligands from damaged hepatocytes. Therefore, TLR3 might be a novel therapeutic target for liver fibrosis. (Hepatology 2016;64:616-631).

Impaired Osteogenesis of Disease-Specific Induced Pluripotent Stem Cells Derived from a CFC Syndrome Patient.

Cardiofaciocutaneous (CFC) syndrome is a rare genetic disorder caused by mutations in the extracellular signal-regulated kinase (ERK) signaling. However, little is known about how aberrant ERK signaling is associated with the defective bone development manifested in most CFC syndrome patients. In this study, induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts of a CFC syndrome patient having rapidly accelerated fibrosarcoma kinase B (BRAF) gain-of-function mutation. CFC-iPSCs were differentiated into mesenchymal stem cells (CFC-MSCs) and further induced to osteoblasts in vitro. The osteogenic defects of CFC-MSCs were revealed by alkaline phosphatase activity assay, mineralization assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. Osteogenesis of CFC-MSCs was attenuated compared to wild-type (WT)-MSCs. In addition to activated ERK signaling, increased p-SMAD2 and decreased p-SMAD1 were observed in CFC-MSCs during osteogenesis. The defective osteogenesis of CFC-MSCs was rescued by inhibition of ERK signaling and SMAD2 signaling or activation of SMAD1 signaling. Importantly, activation of ERK signaling and SMAD2 signaling or inhibition of SMAD1 signaling recapitulated the impaired osteogenesis in WT-MSCs. Our findings indicate that SMAD2 signaling and SMAD1 signaling as well as ERK signaling are responsible for defective early bone development in CFC syndrome, providing a novel insight on the pathological mechanism and therapeutic targets.

Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes.

A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD(+)/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver.

Comparative receptor tyrosine kinase profiling identifies a novel role for AXL in human stem cell pluripotency.

The extensive molecular characterization of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) is required before they can be applied in the future for personalized medicine and drug discovery. Despite the efforts that have been made with kinome analyses, we still lack in-depth insights into the molecular signatures of receptor tyrosine kinases (RTKs) that are related to pluripotency. Here, we present the first detailed and distinct repertoire of RTK characteristic for hPSC pluripotency by determining both the expression and phosphorylation profiles of RTKs in hESCs and hiPSCs using reverse transcriptase-polymerase chain reaction with degenerate primers that target conserved tyrosine kinase domains and phospho-RTK array, respectively. Among the RTKs tested, the up-regulation of EPHA1, ERBB2, FGFR4 and VEGFR2 and the down-regulation of AXL, EPHA4, PDGFRB and TYRO3 in terms of both their expression and phosphorylation levels were predominantly related to the maintenance of hPSC pluripotency. Notably, the specific inhibition of AXL was significantly advantageous in maintaining undifferentiated hESCs and hiPSCs and for the overall efficiency and kinetics of hiPSC generation. Additionally, a global phosphoproteomic analysis showed that ∼30% of the proteins (293 of 970 phosphoproteins) showed differential phosphorylation upon AXL inhibition in undifferentiated hPSCs, revealing the potential contribution of AXL-mediated phosphorylation dynamics to pluripotency-related signaling networks. Our findings provide a novel molecular signature of AXL in pluripotency control that will complement existing pluripotency-kinome networks.

Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs.

Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, ß-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the ß-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wild-type iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered ß-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development.

H2B monoubiquitylation is a 5'-enriched active transcription mark and correlates with exon-intron structure in human cells.

H2B monoubiquitylation (H2Bub1), which is required for multiple methylations of both H3K4 and H3K79, has been implicated in gene expression in numerous organisms ranging from yeast to human. However, the molecular crosstalk between H2Bub1 and other modifications, especially the methylations of H3K4 and H3K79, remains unclear in vertebrates. To better understand the functional role of H2Bub1, we measured genome-wide histone modification patterns in human cells. Our results suggest that H2Bub1 has dual roles, one that is H3 methylation dependent, and another that is H3 methylation independent. First, H2Bub1 is a 5'-enriched active transcription mark and co-occupies with H3K79 methylations in actively transcribed regions. Second, this study shows for the first time that H2Bub1 plays a histone H3 methylations-independent role in chromatin architecture. Furthermore, the results of this work indicate that H2Bub1 is largely positioned at the exon-intron boundaries of highly expressed exons, and it demonstrates increased occupancy in skipped exons compared with flanking exons in the human and mouse genomes. Our findings collectively suggest that a potentiating mechanism links H2Bub1 to both H3K79 methylations in actively transcribed regions and the exon-intron structure of highly expressed exons via the regulation of nucleosome dynamics during transcription elongation.

Autophagy regulates homeostasis of pluripotency-associated proteins in hESCs.

The pluripotency of embryonic stem cells (ESCs) is maintained by intracellular networks of many pluripotency-associated (PA) proteins such as OCT4, SOX2, and NANOG. However, the mechanisms underlying the regulation of protein homeostasis for pluripotency remain elusive. Here, we first demonstrate that autophagy acts together with the ubiquitin-proteasome system (UPS) to modulate the levels of PA proteins in human ESCs (hESCs). Autophagy inhibition impaired the pluripotency despite increment of PA proteins in hESCs. Immunogold-electron microscopy confirmed localization of OCT4 molecules within autophagosomes. Also, knockdown of LC3 expression led to accumulation of PA proteins and reduction of pluripotency in hESCs. Interestingly, autophagy and the UPS showed differential kinetics in the degradation of PA proteins. Autophagy inhibition caused enhanced accumulation of both cytoplasmic and nuclear PA proteins, whereas the UPS inhibition led to preferentially degrade nuclear PA proteins. Our findings suggest that autophagy modulates homeostasis of PA proteins, providing a new insight in the regulation of pluripotency in hESCs.

Modeling of Menkes disease via human induced pluripotent stem cells.

Menkes disease (MD) is a copper-deficient neurodegenerative disorder that manifests severe neurologic symptoms such as seizures, lethargic states, and hypotonia. Menkes disease is due to a dysfunction of ATP7A, but the pathophysiology of neurologic manifestation is poorly understood during embryonic development. To understand the pathophysiology of neurologic symptoms, molecular and cellular phenotypes were investigated in Menkes disease-derived induced pluripotent stem cells (MD-iPSCs). MD-iPSCs were generated from fibroblasts of a Menkes disease patient. Abnormal reticular distribution of ATP7A was observed in MD-fibroblasts and MD-iPSCs, respectively. MD-iPSCs showed abnormal morphology in appearance during embryoid body (EB) formation as compared with wild type (WT)-iPSCs. Intriguingly, aberrant switch of E-cadherin (E-cad) to N-cadherin (N-cad) and impaired neural rosette formation were shown in MD-iPSCs during early differentiation. When extracellular copper was chelated in WT-iPSCs by treatment with bathocuprione sulfate, aberrant switch of E-cad to N-cad and impaired neuronal differentiation were observed, like in MD-iPSCs. Our results suggest that neurological defects in Menkes disease patients may be responsible for aberrant cadherin transition and impaired neuronal differentiation during early developmental stage.

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro.

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.

Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency.

Reduced expression 1 (REX1) is a widely used pluripotency marker, but little is known about its roles in pluripotency. Here, we show that REX1 is functionally important in the reacquisition and maintenance of pluripotency. REX1-depleted human pluripotent stem cells (hPSCs) lose their self-renewal capacity and full differentiation potential, especially their mesoderm lineage potential. Cyclin B1/B2 expression was found to parallel that of REX1. REX1 positively regulates the transcriptional activity of cyclin B1/B2 through binding to their promoters. REX1 induces the phosphorylation of DRP1 at Ser616 by cyclin B/CDK1, which leads to mitochondrial fission and appears to be important for meeting the high-energy demands of highly glycolytic hPSCs. During reprogramming to pluripotency by defined factors (OCT4, SOX2, KLF4, and c-MYC), the reprogramming kinetics and efficiency are markedly improved by adding REX1 or replacing KLF4 with REX1. These improvements are achieved by lowering reprogramming barriers (growth arrest and apoptosis), by enhancing mitochondrial fission, and by conversion to glycolytic metabolism, dependent on the cyclin B1/B2-DRP1 pathway. Our results show that a novel pluripotency regulator, REX1, is essential for pluripotency and reprogramming.

Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway.

Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.

Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

Human histone H3K79 methyltransferase DOT1L protein [corrected] binds actively transcribing RNA polymerase II to regulate gene expression.

Histone-modifying enzymes play a pivotal role in gene expression and repression. In human, DOT1L (Dot1-like) is the only known histone H3 lysine 79 methyltransferase. hDOT1L is associated with transcriptional activation, but the general mechanism connecting hDOT1L to active transcription remains largely unknown. Here, we report that hDOT1L interacts with the phosphorylated C-terminal domain of actively transcribing RNA polymerase II (RNAPII) through a region conserved uniquely in multicellular DOT1 proteins. Genome-wide profiling analyses indicate that the occupancy of hDOT1L largely overlaps with that of RNAPII at actively transcribed genes, especially surrounding transcriptional start sites, in embryonic carcinoma NCCIT cells. We also find that C-terminal domain binding or H3K79 methylations by hDOT1L is important for the expression of target genes such as NANOG and OCT4 and a marker for pluripotency in NCCIT cells. Our results indicate that a functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to actively transcribed genes.

Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro.

Embryonic stem cells (ESCs) maintain unique epigenetic states to maintain their pluripotency. Differentiation of ESCs into specialized cell types requires changes in these epigenetic states. However, the dynamics of epigenetic marks found in hESCs during differentiation are poorly understood. Here, we report the variation in the dynamics of epigenetic modifications associated with the expression of lineage-specific genes during differentiation of hESCs to hepatocytes in vitro. The promoter regions of pluripotency marker genes characterized by permissive histone marks such as trimethylation of H3 at lysine 4 (H3K4me3) and acetylation of H3 at lysine 9 (H3K9ac) in hESCs were instead enriched with repressive histone marks such as dimethylation of H3 at lysine 9 (H3K9me2), trimethylation of H3 at lysine 9 (H3K9me3) and trimethylation of H3 at lysine 27 (H3K27me3) during differentiation to hepatocytes. Interestingly, expression of definitive endoderm marker genes containing bivalent and non-bivalent domains may be modulated by a marked reduction in H3K27me3 and a significant enhancement of permissive marks such as H3K4me3 and H3K9ac during hESC differentiation. Expression of hepatocyte marker genes regulated by histone modifications was similar to that of pluripotency marker genes. Our findings provide insight into the epigenetic mechanisms regulating expression of developmental genes. Of particular interest, they may be differentially regulated either in a bivalent or non-bivalent domain manner during hESC differentiation.