Publications by authors named "Stephen Dalton"

87 Publications

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

Am J Hum Genet 2021 Jun 7;108(6):1040-1052. Epub 2021 May 7.

Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.013DOI Listing
June 2021

Replication timing maintains the global epigenetic state in human cells.

Science 2021 04 22;372(6540):371-378. Epub 2021 Apr 22.

Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.
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http://dx.doi.org/10.1126/science.aba5545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173839PMC
April 2021

Multipotent Vascular Progenitor Cells of the Mesothelium Lineage Generated from Human Pluripotent Stem Cells.

STAR Protoc 2020 Jun 3;1(1):100031. Epub 2020 Jun 3.

Center for Molecular Medicine, 325 Riverbend Road, Athens, GA 30605, USA.

Vascularization is critical for organ homeostasis and function, but cell-based technologies that promote vascular regeneration are limited. This protocol describes steps to generate human pluripotent stem cell (hPSC)-derived vascular progenitors of the mesothelium lineage. This technology has several advantages for the generation of vascular cells. First and foremost, MesoT cells are multipotent progenitors that can generate smooth muscle cells and endothelial cells. MesoT cells therefore have potential utility in tissue repair, tissue engineering, and in vascularization of laboratory grown organs. For complete details on the use and execution of this protocol, please refer to Colunga et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580083PMC
June 2020

A Qualitative Change in the Transcriptome Occurs after the First Cell Cycle and Coincides with Lumen Establishment during MDCKII Cystogenesis.

iScience 2020 Oct 29;23(10):101629. Epub 2020 Sep 29.

Department of Biochemistry and Molecular Biology, Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA.

Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of β-catenin, supported by analyses with - and -knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis.
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http://dx.doi.org/10.1016/j.isci.2020.101629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567049PMC
October 2020

Generation of Functional Brown Adipocytes from Human Pluripotent Stem Cells via Progression through a Paraxial Mesoderm State.

Cell Stem Cell 2020 11 11;27(5):784-797.e11. Epub 2020 Aug 11.

Center for Molecular Medicine, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA; Department of Biochemistry and Molecular Biology, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA. Electronic address:

Brown adipocytes (BAs) are a potential cell source for the treatment of metabolic disease, including type 2 diabetes. In this report, human pluripotent stem cells (hPSCs) are subject to directed differentiation through a paraxial mesoderm progenitor state that generates BAs at high efficiency. Molecular analysis identifies potential regulatory networks for BA development, giving insight into development along this lineage. hPSC-derived BAs undergo elevated rates of glycolysis, uncoupled respiration, and lipolysis that are responsive to changes in cyclic AMP (cAMP)-dependent signaling, consistent with metabolic activity in BA tissue depots. Transplanted human BAs engraft into the inter-scapular region of recipient mice and exhibit thermogenic activity. Recipient animals have elevated metabolic activity, respiratory exchange ratios, and whole-body energy expenditure. Finally, transplanted BAs reduce circulating glucose levels in hyperglycemic animals. These data provide a roadmap for brown adipocyte development and indicate that BAs generated from hPSCs have potential as a tool for therapeutic development.
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http://dx.doi.org/10.1016/j.stem.2020.07.013DOI Listing
November 2020

Landscape of cohesin-mediated chromatin loops in the human genome.

Nature 2020 07 29;583(7818):737-743. Epub 2020 Jul 29.

Department of Genetics, Stanford University School of Medicine, Palo Alto, CA, USA.

Physical interactions between distal regulatory elements have a key role in regulating gene expression, but the extent to which these interactions vary between cell types and contribute to cell-type-specific gene expression remains unclear. Here, to address these questions as part of phase III of the Encyclopedia of DNA Elements (ENCODE), we mapped cohesin-mediated chromatin loops, using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), and analysed gene expression in 24 diverse human cell types, including core ENCODE cell lines. Twenty-eight per cent of all chromatin loops vary across cell types; these variations modestly correlate with changes in gene expression and are effective at grouping cell types according to their tissue of origin. The connectivity of genes corresponds to different functional classes, with housekeeping genes having few contacts, and dosage-sensitive genes being more connected to enhancer elements. This atlas of chromatin loops complements the diverse maps of regulatory architecture that comprise the ENCODE Encyclopedia, and will help to support emerging analyses of genome structure and function.
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http://dx.doi.org/10.1038/s41586-020-2151-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410831PMC
July 2020

Human beige adipocytes for drug discovery and cell therapy in metabolic diseases.

Nat Commun 2020 06 2;11(1):2758. Epub 2020 Jun 2.

Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, University of Georgia, Athens, GA, 30602, USA.

Human beige adipocytes (BAs) have potential utility for the development of therapeutics to treat diabetes and obesity-associated diseases. Although several reports have described the generation of beige adipocytes in vitro, their potential utility in cell therapy and drug discovery has not been reported. Here, we describe the generation of BAs from human adipose-derived stem/stromal cells (ADSCs) in serum-free medium with efficiencies >90%. Molecular profiling of beige adipocytes shows them to be similar to primary BAs isolated from human tissue. In vitro, beige adipocytes exhibit uncoupled mitochondrial respiration and cAMP-induced lipolytic activity. Following transplantation, BAs increase whole-body energy expenditure and oxygen consumption, while reducing body-weight in recipient mice. Finally, we show the therapeutic utility of BAs in a platform for high-throughput drug screening (HTS). These findings demonstrate the potential utility of BAs as a cell therapeutic and as a tool for the identification of drugs to treat metabolic diseases.
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http://dx.doi.org/10.1038/s41467-020-16340-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265435PMC
June 2020

Role of cyclins and cyclin-dependent kinases in pluripotent stem cells and their potential as a therapeutic target.

Semin Cell Dev Biol 2020 11 14;107:63-71. Epub 2020 May 14.

Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand. Electronic address:

Over the last 15 years connections between cell cycle control, maintenance of pluripotency, and control of cell fate decisions have been firmly established. With the emergence of powerful tools, such as highly-specific small molecule inhibitors for cyclin-dependent protein kinase (CDK) activity and single-cell imaging technologies, the mechanistic links between cyclins, CDKs and regulation in PSCs in mechanistic detail has been made possible. In this review, we discuss new developments that mechanistically link the CDK regulatory network to control of cell fate decisions, including maintenance of the pluripotent state. Overall, these findings have potential to impact the translational applications of stem cells in regenerative medicine, drug discovery and cancer treatment.
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http://dx.doi.org/10.1016/j.semcdb.2020.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554155PMC
November 2020

Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux.

Stem Cells Int 2019 19;2019:8195614. Epub 2019 May 19.

School of BioSciences, The University of Melbourne, 11 Royal Parade, Parkville, 3010 VIC, Australia.

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.
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http://dx.doi.org/10.1155/2019/8195614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545818PMC
May 2019

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

Stem Cell Reports 2019 07 20;13(1):193-206. Epub 2019 Jun 20.

Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, USA. Electronic address:

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.
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http://dx.doi.org/10.1016/j.stemcr.2019.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627004PMC
July 2019

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair.

Cell Rep 2019 03;26(10):2566-2579.e10

Department of Biochemistry and Molecular Biology and Center for Molecular Medicine, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA. Electronic address:

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.
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http://dx.doi.org/10.1016/j.celrep.2019.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585464PMC
March 2019

Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis.

Oncotarget 2018 Jun 26;9(49):29162-29179. Epub 2018 Jun 26.

Department of Biochemistry and Molecular Biology, Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA.

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, , a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.
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http://dx.doi.org/10.18632/oncotarget.25646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044369PMC
June 2018

Building Blood Vessels with Vascular Progenitor Cells.

Trends Mol Med 2018 07 22;24(7):630-641. Epub 2018 May 22.

Center for Molecular Medicine, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA; Department of Biochemistry and Molecular Biology, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA. Electronic address:

Vascular progenitor cells have been identified from perivascular cell fractions and peripheral blood and bone marrow mononuclear fractions. These vascular progenitors share the ability to generate some of the vascular lineages, including endothelial cells, smooth muscle cells, and pericytes. The potential therapeutic uses for vascular progenitor cells are broad and relate to stroke, ischemic disease, and to the engineering of whole organs and tissues that require a vascular component. This review summarizes the best-characterized sources of vascular progenitor cells and discusses advances in 3D printing and electrospinning using blended polymers for the creation of biomimetic vascular grafts. These advances are pushing the field of regenerative medicine closer to the creation of small-diameter vascular grafts with long-term clinical utility.
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http://dx.doi.org/10.1016/j.molmed.2018.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050017PMC
July 2018

What Can 'Brown-ing' Do For You?

Trends Endocrinol Metab 2018 05 29;29(5):349-359. Epub 2018 Mar 29.

Center for Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Georgia, 325 Riverbend Road, Athens, GA 30602, USA. Electronic address:

Human stem cell-based models of thermogenic adipocytes provide an opportunity for the establishment of new therapeutics, modeling of disease mechanisms, and understanding of development. Pluripotent stem cells, adipose-derived stem cells/preadipocytes, and programming-reprogramming-based approaches have been used to develop cell-based platforms for drug screening and transplantable therapeutics in the metabolic disease arena. Here we provide a detailed overview of these approaches, the latest advances in this field, and the opportunities and shortcomings they present. Moreover, we comment on how stem-cell-based platforms can be best utilized in the future for the treatment and understanding of metabolic diseases, including type 2 diabetes and associated medical issues such as obesity.
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http://dx.doi.org/10.1016/j.tem.2018.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937921PMC
May 2018

Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency.

Cell Stem Cell 2017 10;21(4):449-455.e4

Department of Molecular Biology, Faculty of Science, Radboud University, 6525GA Nijmegen, the Netherlands. Electronic address:

Mouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.
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http://dx.doi.org/10.1016/j.stem.2017.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658514PMC
October 2017

MYC Controls Human Pluripotent Stem Cell Fate Decisions through Regulation of Metabolic Flux.

Cell Stem Cell 2017 10 28;21(4):502-516.e9. Epub 2017 Sep 28.

Department of Biochemistry and Molecular Biology, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA; Center for Molecular Medicine, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Electronic address:

As human pluripotent stem cells (hPSCs) exit pluripotency, they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that, although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and, in fact, essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiation coincides with a reduction in MYC/MYCN and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of "switch" genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.
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http://dx.doi.org/10.1016/j.stem.2017.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644510PMC
October 2017

Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development.

Curr Opin Genet Dev 2017 Oct 4;46:44-49. Epub 2017 Jul 4.

Department of Biochemistry and Molecular Biology and Center for Molecular Medicine, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Electronic address:

Cell fate decisions are closely linked to changes in metabolic activity. Over recent years this connection has been implicated in mechanisms underpinning embryonic development, reprogramming and disease pathogenesis. In addition to being important for supporting the energy demands of different cell types, metabolic switching from aerobic glycolysis to oxidative phosphorylation plays a critical role in controlling biosynthetic processes, intracellular redox state, epigenetic status and reactive oxygen species levels. These processes extend beyond ATP synthesis by impacting cell proliferation, differentiation, enzymatic activity, ageing and genomic integrity. This review will focus on how metabolic switching impacts decisions made by multipotent cells and discusses mechanisms by which this occurs.
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http://dx.doi.org/10.1016/j.gde.2017.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842063PMC
October 2017

Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming.

Development 2016 12;143(23):4301-4311

Center for Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA

A strong connection exists between the cell cycle and mechanisms required for executing cell fate decisions in a wide-range of developmental contexts. Terminal differentiation is often associated with cell cycle exit, whereas cell fate switches are frequently linked to cell cycle transitions in dividing cells. These phenomena have been investigated in the context of reprogramming, differentiation and trans-differentiation but the underpinning molecular mechanisms remain unclear. Most progress to address the connection between cell fate and the cell cycle has been made in pluripotent stem cells, in which the transition through mitosis and G1 phase is crucial for establishing a window of opportunity for pluripotency exit and the initiation of differentiation. This Review will summarize recent developments in this area and place them in a broader context that has implications for a wide range of developmental scenarios.
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http://dx.doi.org/10.1242/dev.142075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201050PMC
December 2016

Glycosylation and stem cells: Regulatory roles and application of iPSCs in the study of glycosylation-related disorders.

Bioessays 2016 12 26;38(12):1255-1265. Epub 2016 Sep 26.

Department of Biochemistry and Molecular Biology, Athens, GA, USA.

Glycosylation refers to the co- and post-translational modification of protein and lipids by monosaccharides or oligosaccharide chains. The surface of mammalian cells is decorated by a heterogeneous and highly complex array of protein and lipid linked glycan structures that vary significantly between different cell types, raising questions about their roles in development and disease pathogenesis. This review will begin by focusing on recent findings that define roles for cell surface protein and lipid glycosylation in pluripotent stem cells and their functional impact during normal development. Then, we will describe how patient derived induced pluripotent stem cells are being used to model human diseases such as congenital disorders of glycosylation. Collectively, these studies indicate that cell surface glycans perform critical roles in human development and disease.
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http://dx.doi.org/10.1002/bies.201600138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214967PMC
December 2016

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS.

J Proteome Res 2016 09 3;15(9):3196-203. Epub 2016 Aug 3.

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, United States.

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.
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http://dx.doi.org/10.1021/acs.jproteome.6b00414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261780PMC
September 2016

A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity.

Nat Commun 2016 06 15;7:11903. Epub 2016 Jun 15.

Department of Epigenetics, Fondazione Istituto Nazionale di Genetica Molecolare 'Romeo ed Enrica Invernizzi', Via Francesco Sforza 35, Milan 20122, Italy.

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.
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http://dx.doi.org/10.1038/ncomms11903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912626PMC
June 2016

Nucleosome positioning changes during human embryonic stem cell differentiation.

Epigenetics 2016 06 18;11(6):426-37. Epub 2016 Apr 18.

a Department of Biochemistry and Molecular Biology , Institute of Bioinformatics, University of Georgia , Athens , GA , USA.

Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content.
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http://dx.doi.org/10.1080/15592294.2016.1176649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939925PMC
June 2016

ST8SIA4-Dependent Polysialylation is Part of a Developmental Program Required for Germ Layer Formation from Human Pluripotent Stem Cells.

Stem Cells 2016 07 3;34(7):1742-52. Epub 2016 May 3.

Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia, USA.

Polysialic acid (PSA) is a carbohydrate polymer of repeating α-2,8 sialic acid residues that decorates multiple targets, including neural cell adhesion molecule (NCAM). PST and STX encode the two enzymes responsible for PSA modification of target proteins in mammalian cells, but despite widespread polysialylation in embryonic development, the majority of studies have focused strictly on the role of PSA in neurogenesis. Using human pluripotent stem cells (hPSCs), we have revisited the developmental role of PST and STX and show that early progenitors of the three embryonic germ layers are polysialylated on their cell surface. Changes in polysialylation can be attributed to lineage-specific expression of polysialyltransferase genes; PST is elevated in endoderm and mesoderm, while STX is elevated in ectoderm. In hPSCs, PST and STX genes are epigenetically marked by overlapping domains of H3K27 and H3K4 trimethylation, indicating that they are held in a "developmentally-primed" state. Activation of PST transcription during early mesendoderm differentiation is under control of the T-Goosecoid transcription factor network, a key regulatory axis required for early cell fate decisions in the vertebrate embryo. This establishes polysialyltransferase genes as part of a developmental program associated with germ layer establishment. Finally, we show by shRNA knockdown and CRISPR-Cas9 genome editing that PST-dependent cell surface polysialylation is essential for endoderm specification. This is the first report to demonstrate a role for a glycosyltransferase in hPSC lineage specification. Stem Cells 2016;34:1742-1752.
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http://dx.doi.org/10.1002/stem.2379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931981PMC
July 2016

Concise Review: Control of Cell Fate Through Cell Cycle and Pluripotency Networks.

Stem Cells 2016 06 16;34(6):1427-36. Epub 2016 Mar 16.

Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, Athens, Georgia, USA.

Pluripotent stem cells (PSCs) proliferate rapidly with a characteristic cell cycle structure consisting of short G1- and G2-gap phases. This applies broadly to PSCs of peri-implantation stage embryos, cultures of embryonic stem cells, induced pluripotent stem cells, and embryonal carcinoma cells. During the early stages of PSC differentiation however, cell division times increase as a consequence of cell cycle remodeling. Most notably, this is indicated by elongation of the G1-phase. Observations linking changes in the cell cycle with exit from pluripotency have raised questions about the role of cell cycle control in maintenance of the pluripotent state. Until recently however, this has been a difficult question to address because of limitations associated with experimental tools. Recent studies now show that pluripotency and cell cycle regulatory networks are intertwined and that cell cycle control mechanisms are an integral, mechanistic part of the PSC state. Studies in embryonal carcinoma, some 30 years ago, first suggested that pluripotent cells initiate differentiation when in the G1-phase. More recently, a molecular "priming" mechanism has been proposed to explain these observations in human embryonic stem cells. Complexity in this area has been increased by the realization that pluripotent cells exist in multiple developmental states and that in addition to each having their own characteristic gene expression and epigenetic signatures, they potentially have alternate modes of cell cycle regulation. This review will summarize current knowledge in these areas and will highlight important aspects of interconnections between the cell cycle, self-renewal, pluripotency, and cell fate decisions. Stem Cells 2016;34:1427-1436.
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http://dx.doi.org/10.1002/stem.2345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201256PMC
June 2016

Metabolic Reprogramming of Stem Cell Epigenetics.

Cell Stem Cell 2015 Dec;17(6):651-662

Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20829, USA. Electronic address:

For many years, stem cell metabolism was viewed as a byproduct of cell fate status rather than an active regulatory mechanism; however, there is now a growing appreciation that metabolic pathways influence epigenetic changes associated with lineage commitment, specification, and self-renewal. Here we review how metabolites generated during glycolytic and oxidative processes are utilized in enzymatic reactions leading to epigenetic modifications and transcriptional regulation. We discuss how "metabolic reprogramming" contributes to global epigenetic changes in the context of naive and primed pluripotent states, somatic reprogramming, and hematopoietic and skeletal muscle tissue stem cells, and we discuss the implications for regenerative medicine.
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http://dx.doi.org/10.1016/j.stem.2015.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672395PMC
December 2015

Long Noncoding RNA ADINR Regulates Adipogenesis by Transcriptionally Activating C/EBPα.

Stem Cell Reports 2015 Nov 17;5(5):856-865. Epub 2015 Oct 17.

Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Non-coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address:

C/EBPα is a critical transcriptional regulator of adipogenesis. How C/EBPα transcription is itself regulated is poorly understood, however, and remains a key question that needs to be addressed for a complete understanding of adipogenic development. Here, we identify a lncRNA, ADINR (adipogenic differentiation induced noncoding RNA), transcribed from a position ∼450 bp upstream of the C/EBPα gene, that orchestrates C/EBPα transcription in vivo. Depletion of ADINR leads to a severe adipogenic defect that is rescued by overexpression of C/EBPα. Moreover, we reveal that ADINR RNA specifically binds to PA1 and recruits MLL3/4 histone methyl-transferase complexes so as to increase H3K4me3 and decrease H3K27me3 histone modification in the C/EBPα locus during adipogenesis. These results show that ADINR plays important roles in regulating the differentiation of human mesenchymal stem cells into adipocytes by modulating C/EBPα in cis.
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http://dx.doi.org/10.1016/j.stemcr.2015.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649137PMC
November 2015

Linking the Cell Cycle to Cell Fate Decisions.

Authors:
Stephen Dalton

Trends Cell Biol 2015 Oct;25(10):592-600

Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, 500 DW Brooks Drive, Athens, GA 30602, USA. Electronic address:

Pluripotent stem cells (PSCs) retain the ability to differentiate into a wide range of cell types while undergoing self-renewal. They also exhibit an unusual mode of cell cycle regulation, reflected by a cell cycle structure where G1 and G2 phases are truncated. When individual PSCs are exposed to specification cues, they activate developmental programs and remodel the cell cycle so that the length of G1 and overall cell division times increase. The response of individual stem cells to pro-differentiation signals is strikingly heterogeneous, resulting in asynchronous differentiation. Recent evidence indicates that this phenomenon is due to cell cycle-dependent mechanisms that restrict the initial activation of developmental genes to the G1 phase. This suggests a broad biological mechanism where multipotent cells are 'primed' to initiate cell fate decisions during their transition through G1. Here, I discuss mechanisms underpinning the commitment towards the differentiated state and its relation to the cell cycle.
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http://dx.doi.org/10.1016/j.tcb.2015.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4584407PMC
October 2015

Utilizing FUCCI reporters to understand pluripotent stem cell biology.

Methods 2016 05 21;101:4-10. Epub 2015 Sep 21.

Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Electronic address:

The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs, elutriation and temperature sensitive mutants. Importantly, FUCCI reporters allow cell cycle events in dynamic systems, such as differentiation, to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report, we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible.
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http://dx.doi.org/10.1016/j.ymeth.2015.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801677PMC
May 2016

Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency.

Stem Cell Reports 2015 Sep 13;5(3):323-36. Epub 2015 Aug 13.

Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Electronic address:

Here we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a "window of opportunity" for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase.
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http://dx.doi.org/10.1016/j.stemcr.2015.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618451PMC
September 2015