Publications by authors named "Effie Apostolou"

36 Publications

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture.

Nature 2021 01 9;589(7841):299-305. Epub 2020 Dec 9.

Division of Hematology and Medical Oncology, Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.
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http://dx.doi.org/10.1038/s41586-020-3017-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855728PMC
January 2021

Dynamic 3D Chromatin Reorganization during Establishment and Maintenance of Pluripotency.

Stem Cell Reports 2020 12 25;15(6):1176-1195. Epub 2020 Nov 25.

Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Higher-order chromatin structure is tightly linked to gene expression and therefore cell identity. In recent years, the chromatin landscape of pluripotent stem cells has become better characterized, and unique features at various architectural levels have been revealed. However, the mechanisms that govern establishment and maintenance of these topological characteristics and the temporal and functional relationships with transcriptional or epigenetic features are still areas of intense study. Here, we will discuss progress and limitations of our current understanding regarding how the 3D chromatin topology of pluripotent stem cells is established during somatic cell reprogramming and maintained during cell division. We will also discuss evidence and theories about the driving forces of topological reorganization and the functional links with key features and properties of pluripotent stem cell identity.
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http://dx.doi.org/10.1016/j.stemcr.2020.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724465PMC
December 2020

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

Stem Cell Reports 2020 12 24;15(6):1233-1245. Epub 2020 Sep 24.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU Langone Medical Center, New York, NY 10016, USA; Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU Langone Medical Center, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY 10016, USA; Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
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http://dx.doi.org/10.1016/j.stemcr.2020.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724475PMC
December 2020

Transcription factors: building hubs in the 3D space.

Cell Cycle 2020 Oct 12;19(19):2395-2410. Epub 2020 Aug 12.

Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine , New York, NY, USA.

The hierarchical three-dimensional folding of the mammalian genome constitutes an important regulatory layer of gene expression and cell fate control during processes such as development and tumorigenesis. Accumulating evidence supports the existence of complex topological assemblies in which multiple genes and regulatory elements are frequently interacting with each other in the 3D nucleus. Here, we will discuss the nature, organizational principles, and potential function of such assemblies, including the recently reported enhancer "hubs," "cliques," and FIREs (frequently interacting regions) as well as multi-contact hubs. We will also review recent studies that investigate the role of transcription factors (TFs) in driving the topological genome reorganization and hub formation in the context of cell fate transitions and cancer. Finally, we will highlight technological advances that enabled these studies, current limitations, and future directions necessary to advance our understating in the field.
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http://dx.doi.org/10.1080/15384101.2020.1805238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553511PMC
October 2020

A Susceptibility Locus on Chromosome 13 Profoundly Impacts the Stability of Genomic Imprinting in Mouse Pluripotent Stem Cells.

Cell Rep 2020 03;30(11):3597-3604.e3

Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU Langone Medical Center, New York, NY 10016, USA; Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU Langone Medical Center, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY 10016, USA; Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Cultured pluripotent cells accumulate detrimental chromatin alterations, including DNA methylation changes at imprinted genes known as loss of imprinting (LOI). Although the occurrence of LOI is considered a stochastic phenomenon, here we document a genetic determinant that segregates mouse pluripotent cells into stable and unstable cell lines. Unstable lines exhibit hypermethylation at Dlk1-Dio3 and other imprinted loci, in addition to impaired developmental potential. Stimulation of demethylases by ascorbic acid prevents LOI and loss of developmental potential. Susceptibility to LOI greatly differs between commonly used mouse strains, which we use to map a causal region on chromosome 13 with quantitative trait locus (QTL) analysis. Our observations identify a strong genetic determinant of locus-specific chromatin abnormalities in pluripotent cells and provide a non-invasive way to suppress them. This highlights the importance of considering genetics in conjunction with culture conditions for assuring the quality of pluripotent cells for biomedical applications.
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http://dx.doi.org/10.1016/j.celrep.2020.02.073DOI Listing
March 2020

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

Genome Biol 2019 11 21;20(1):248. Epub 2019 Nov 21.

New York University Langone Health, New York, NY, USA.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.
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http://dx.doi.org/10.1186/s13059-019-1853-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868874PMC
November 2019

KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks.

Nat Cell Biol 2019 10 23;21(10):1179-1190. Epub 2019 Sep 23.

Sanford I Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
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http://dx.doi.org/10.1038/s41556-019-0390-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339746PMC
October 2019

Identification of Cancer Drivers at CTCF Insulators in 1,962 Whole Genomes.

Cell Syst 2019 05 8;8(5):446-455.e8. Epub 2019 May 8.

Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA; Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA; Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA; Caryl and Israel Englander Institute for Precision Medicine, New York Presbyterian Hospital, Weill Cornell Medicine, New York, NY 10065, USA. Electronic address:

Recent studies have shown that mutations at non-coding elements, such as promoters and enhancers, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF insulators, has been overlooked in the previous driver analyses. We used insulator annotations from CTCF and cohesin ChIA-PET and analyzed somatic mutations in 1,962 whole genomes from 21 cancer types. Using the heterogeneous patterns of transcription-factor-motif disruption, functional impact, and recurrence of mutations, we developed a computational method that revealed 21 insulators showing signals of positive selection. In particular, mutations in an insulator in multiple cancer types, including 16% of melanoma samples, are associated with TGFB1 up-regulation. Using CRISPR-Cas9, we find that alterations at two of the most frequently mutated regions in this insulator increase cell growth by 40%-50%, supporting the role of this boundary element as a cancer driver. Thus, our study reveals several CTCF insulators as putative cancer drivers.
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http://dx.doi.org/10.1016/j.cels.2019.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917527PMC
May 2019

TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.

Mol Cell 2019 06 17;74(6):1148-1163.e7. Epub 2019 Apr 17.

Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia. Electronic address:

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.
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http://dx.doi.org/10.1016/j.molcel.2019.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671628PMC
June 2019

A hPSC-based platform to discover gene-environment interactions that impact human β-cell and dopamine neuron survival.

Nat Commun 2018 11 16;9(1):4815. Epub 2018 Nov 16.

Department of Surgery, Weill Cornell Medical College, 1300 York Ave, New York, 10065, NY, USA.

Common disorders, including diabetes and Parkinson's disease, are caused by a combination of environmental factors and genetic susceptibility. However, defining the mechanisms underlying gene-environment interactions has been challenging due to the lack of a suitable experimental platform. Using pancreatic β-like cells derived from human pluripotent stem cells (hPSCs), we discovered that a commonly used pesticide, propargite, induces pancreatic β-cell death, a pathological hallmark of diabetes. Screening a panel of diverse hPSC-derived cell types we extended this observation to a similar susceptibility in midbrain dopamine neurons, a cell type affected in Parkinson's disease. We assessed gene-environment interactions using isogenic hPSC lines for genetic variants associated with diabetes and Parkinson's disease. We found GSTT1 pancreatic β-like cells and dopamine neurons were both hypersensitive to propargite-induced cell death. Our study identifies an environmental chemical that contributes to human β-cell and dopamine neuron loss and validates a novel hPSC-based platform for determining gene-environment interactions.
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http://dx.doi.org/10.1038/s41467-018-07201-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240096PMC
November 2018

Cellular trajectories and molecular mechanisms of iPSC reprogramming.

Curr Opin Genet Dev 2018 10 17;52:77-85. Epub 2018 Jun 17.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology and Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology.
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http://dx.doi.org/10.1016/j.gde.2018.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252123PMC
October 2018

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes.

Cell Rep 2018 01 28;22(4):876-884. Epub 2018 Jan 28.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology and Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.
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http://dx.doi.org/10.1016/j.celrep.2017.12.098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664440PMC
January 2018

Shaping the Pluripotent Genome: Switches, Borders, and Loops.

Authors:
Effie Apostolou

Cell Stem Cell 2018 02;22(2):148-150

Joan & Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Three-dimensional genome organization is largely cell type specific and requires reorganization during cell fate transitions. A recent study in Nature Genetics (Stadhouders et al., 2018) offers important insights into the principles and drivers of such reorganization during reprogramming of somatic cells into pluripotent stem cells.
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http://dx.doi.org/10.1016/j.stem.2018.01.005DOI Listing
February 2018

Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells.

Cell Rep 2017 05;19(7):1283-1293

Joan & Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

During mitosis, transcription is halted and many chromatin features are lost, posing a challenge for the continuity of cell identity, particularly in fast cycling stem cells, which constantly balance self-renewal with differentiation. Here we show that, in pluripotent stem cells, certain histone marks and stem cell regulators remain associated with specific genomic regions of mitotic chromatin, a phenomenon known as mitotic bookmarking. Enhancers of stem cell-related genes are bookmarked by both H3K27ac and the master regulators OCT4, SOX2, and KLF4, while promoters of housekeeping genes retain high levels of mitotic H3K27ac in a cell-type invariant manner. Temporal degradation of OCT4 during mitotic exit compromises its ability both to maintain and induce pluripotency, suggesting that its regulatory function partly depends on its bookmarking activity. Together, our data document a widespread yet specific bookmarking by histone modifications and transcription factors promoting faithful and efficient propagation of stemness after cell division.
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http://dx.doi.org/10.1016/j.celrep.2017.04.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495017PMC
May 2017

The Chromatin Signature of Pluripotency: Establishment and Maintenance.

Curr Stem Cell Rep 2016;2:255-262. Epub 2016 Jun 27.

Weill Cornell Medicine, Division of Hematology and Medical Oncology, Sandra and Edward Meyer Cancer Center, 413E 69th Street, Belfer research Building, New York, NY 10021 USA.

The revolutionary discovery that somatic cells can be reprogrammed by a defined set transcription factors to induced pluripotent stem cells (iPSCs) changed dramatically the way we perceive cell fate determination. Importantly, iPSCs, similar to embryo-derived stem cells (ESCs), are characterized by a remarkable developmental plasticity and the capacity to self-renew "indefinitely" under appropriate culture conditions, opening new avenues for personalized therapy and disease modeling. Elucidating the molecular mechanisms that maintain, induce, or alter stem cell identity is crucial for a deeper understanding of cell fate determination and potential translational applications. Intense research over the last 10 years exploiting technological advances in epigenomics and genome editing has unraveled many of the mysteries of pluripotent identity enabling novel and efficient ways to manipulate it for biomedical purposes. In this review, we focus on the chromatin and epigenetic characteristics that distinguish stem cells from somatic cells and their dynamic changes during differentiation and reprogramming.
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http://dx.doi.org/10.1007/s40778-016-0055-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972866PMC
June 2016

Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming.

Cell Stem Cell 2016 05;18(5):611-24

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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 we 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). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we 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. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.
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http://dx.doi.org/10.1016/j.stem.2016.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859942PMC
May 2016

A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2.

Stem Cell Reports 2016 05 3;6(5):704-716. Epub 2016 Mar 3.

Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.
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http://dx.doi.org/10.1016/j.stemcr.2016.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939549PMC
May 2016

PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells.

Cell Rep 2015 Sep 20;12(9):1456-70. Epub 2015 Aug 20.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute (DFCI), Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute (HHMI), Boston, MA 02115, USA. Electronic address:

Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.
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http://dx.doi.org/10.1016/j.celrep.2015.07.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384103PMC
September 2015

Small molecules facilitate rapid and synchronous iPSC generation.

Nat Methods 2014 Nov 24;11(11):1170-6. Epub 2014 Sep 24.

1] Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [4] Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA. [5] Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.
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http://dx.doi.org/10.1038/nmeth.3142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326224PMC
November 2014

Rearranging the chromatin for pluripotency.

Cell Cycle 2014 15;13(2):167-8. Epub 2013 Nov 15.

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine; Harvard Stem Cell Institute; Boston, MA USA; Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology; Harvard University and Harvard Medical School; Cambridge, MA USA.

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http://dx.doi.org/10.4161/cc.27028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906226PMC
December 2014

Chromatin dynamics during cellular reprogramming.

Nature 2013 Oct;502(7472):462-71

1] Massachusetts General Hospital Center for Regenerative Medicine, 185 Cambridge Street, Boston, Massachusetts 02114, USA. [2] Harvard Stem Cell Institute, 1350 Masschusetts Avenue, Cambridge, Massachusetts 02138, USA. [3] Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, USA. [4] Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.

Induced pluripotency is a powerful tool to derive patient-specific stem cells. In addition, it provides a unique assay to study the interplay between transcription factors and chromatin structure. Here, we review the latest insights into chromatin dynamics that are inherent to induced pluripotency. Moreover, we compare and contrast these events with other physiological and pathological processes that involve changes in chromatin and cell state, including germ cell maturation and tumorigenesis. We propose that an integrated view of these seemingly diverse processes could provide mechanistic insights into cell fate transitions in general and might lead to new approaches in regenerative medicine and cancer treatment.
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http://dx.doi.org/10.1038/nature12749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216318PMC
October 2013

Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming.

Cell Stem Cell 2013 Jun 9;12(6):699-712. Epub 2013 May 9.

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA.

The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin "interactome" for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.
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http://dx.doi.org/10.1016/j.stem.2013.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725985PMC
June 2013

A molecular roadmap of reprogramming somatic cells into iPS cells.

Cell 2012 Dec;151(7):1617-32

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA.

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
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http://dx.doi.org/10.1016/j.cell.2012.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608203PMC
December 2012

Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system.

Cell Stem Cell 2012 Dec 25;11(6):783-98. Epub 2012 Oct 25.

Howard Hughes Medical Institute and Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.
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http://dx.doi.org/10.1016/j.stem.2012.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549668PMC
December 2012

The polycomb group protein L3mbtl2 assembles an atypical PRC1-family complex that is essential in pluripotent stem cells and early development.

Cell Stem Cell 2012 Sep 5;11(3):319-32. Epub 2012 Jul 5.

Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA.

L3mbtl2 has been implicated in transcriptional repression and chromatin compaction but its biological function has not been defined. Here we show that disruption of L3mbtl2 results in embryonic lethality with failure of gastrulation. This correlates with compromised proliferation and abnormal differentiation of L3mbtl2(-/-) embryonic stem (ES) cells. L3mbtl2 regulates genes by recruiting a Polycomb Repressive Complex1 (PRC1)-related complex, resembling the previously described E2F6-complex, and including G9A, Hdac1, and Ring1b. The presence of L3mbtl2 at target genes is associated with H3K9 dimethylation, low histone acetylation, and H2AK119 ubiquitination, but the latter is neither dependent on L3mbtl2 nor sufficient for repression. Genome-wide studies revealed that the L3mbtl2-dependent complex predominantly regulates genes not bound by canonical PRC1 and PRC2. However, some developmental regulators are repressed by the combined activity of all three complexes. Together, we have uncovered a highly selective, essential role for an atypical PRC1-family complex in ES cells and early development.
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http://dx.doi.org/10.1016/j.stem.2012.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647456PMC
September 2012

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells.

Cancer Cell Int 2012 Jun 20;12(1):31. Epub 2012 Jun 20.

Department of Medical Oncology, University Hospital of Larissa, Larissa, Greece.

Background: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro.

Methods: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment.

Results: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA.

Conclusions: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.
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http://dx.doi.org/10.1186/1475-2867-12-31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441896PMC
June 2012

Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells.

Nat Genet 2012 Mar 4;44(4):398-405, S1-2. Epub 2012 Mar 4.

Massachusetts General Hospital Center for Regenerative Medicine, Harvard Stem Cell Institute, Boston, Massachusetts, USA.

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor-mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.
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http://dx.doi.org/10.1038/ng.1110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538378PMC
March 2012

Genomic analysis reveals a novel nuclear factor-κB (NF-κB)-binding site in Alu-repetitive elements.

J Biol Chem 2011 Nov 5;286(44):38768-38782. Epub 2011 Sep 5.

Institute of Molecular Biology, Genetics and Biotechnology, Biomedical Research Foundation, Academy of Athens, 4 Soranou Efesiou Street, Athens 11527, Greece. Electronic address:

The transcription factor NF-κB is a critical regulator of immune responses. To determine how NF-κB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-κB target genes in response to virus infection. We discovered that most of the NF-κB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-κB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-κB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-κB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.
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http://dx.doi.org/10.1074/jbc.M111.234161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207430PMC
November 2011

Stem cells: iPS cells under attack.

Nature 2011 Jun 8;474(7350):165-6. Epub 2011 Jun 8.

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http://dx.doi.org/10.1038/474165aDOI Listing
June 2011

Inverse baseline expression pattern of the NEP/neuropeptides and NFκB/proteasome pathways in androgen-dependent and androgen-independent prostate cancer cells.

Cancer Cell Int 2011 May 15;11(1):13. Epub 2011 May 15.

Department of Medical Oncology, University Hospital of Larissa, Larissa, Greece.

Background: Castration-resistance in prostate cancer (PC) is a critical event hallmarking a switch to a more aggressive phenotype. Neuroendocrine differentiation and upregulation of NFκB transcriptional activity are two mechanisms that have been independently linked to this process.

Methods: We investigated these two pathways together using in vitro models of androgen-dependent (AD) and androgen-independent (AI) PC. We measured cellular levels, activity and surface expression of Neutral Endopeptidase (NEP), levels of secreted Endothelin-1 (ET-1), levels, sub-cellular localisation and DNA binding ability of NFκB, and proteasomal activity in human native PC cell lines (LnCaP and PC-3) modelling AD and AI states.

Results: At baseline, AD cells were found to have high NEP expression and activity and low secreted ET-1. In contrast, they exhibited a low-level activation of the NFκB pathway associated with comparatively low 20S proteasome activity. The AI cells showed the exact mirror image, namely increased proteasomal activity resulting in a canonical pathway-mediated NFκB activation, and minimal NEP activity with increased levels of secreted ET-1.

Conclusions: Our results seem to support evidence for divergent patterns of expression of the NFκB/proteasome pathway with relation to components of the NEP/neuropeptide axis in PC cells of different level of androgen dependence. NEP and ET-1 are inversely and directly related to an activated state of the NFκB/proteasome pathway, respectively. A combination therapy targeting both pathways may ultimately prove to be of benefit in clinical practice.
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http://dx.doi.org/10.1186/1475-2867-11-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121665PMC
May 2011