Publications by authors named "Konstantinos Anastassiadis"

79 Publications

is required for spermatid morphogenesis and male fertility in mice.

Development 2021 Mar 25. Epub 2021 Mar 25.

Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama, Japan

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, , plays an essential role in spermatid morphogenesis. Loss of in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. depletion leads to dysregulation of thousands of genes, including the X chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, is a KMT2B target that is vitally necessary for spermiogenesis and fertility.
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http://dx.doi.org/10.1242/dev.196212DOI Listing
March 2021

Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

Mol Cell 2021 Mar 17. Epub 2021 Mar 17.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen 2200, Denmark. Electronic address:

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.
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http://dx.doi.org/10.1016/j.molcel.2021.03.008DOI Listing
March 2021

A Novel GATA2 Protein Reporter Mouse Reveals Hematopoietic Progenitor Cell Types.

Stem Cell Reports 2020 08 9;15(2):326-339. Epub 2020 Jul 9.

Department of Biosystems Science & Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address:

The transcription factor (TF) GATA2 plays a key role in organ development and cell fate control in the central nervous, urogenital, respiratory, and reproductive systems, and in primitive and definitive hematopoiesis. Here, we generate a knockin protein reporter mouse line expressing a GATA2VENUS fusion from the endogenous Gata2 genomic locus, with correct expression and localization of GATA2VENUS in different organs. GATA2VENUS expression is heterogeneous in different hematopoietic stem and progenitor cell populations (HSPCs), identifies functionally distinct subsets, and suggests a novel monocyte and mast cell lineage bifurcation point. GATA2 levels further correlate with proliferation and lineage outcome of hematopoietic progenitors. The GATA2VENUS mouse line improves the identification of specific live cell types during embryonic and adult development and will be crucial for analyzing GATA2 protein dynamics in TF networks.
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http://dx.doi.org/10.1016/j.stemcr.2020.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419669PMC
August 2020

MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation.

Development 2020 06 17;147(12). Epub 2020 Jun 17.

Genomics, Center for Molecular and Cellular Bioengineering, Biotechnology Center, Technische Universität Dresden, Tatzberg 47, 01307 Dresden, Germany

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of and mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.
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http://dx.doi.org/10.1242/dev.186999DOI Listing
June 2020

The role of SETD1A and SETD1B in development and disease.

Biochim Biophys Acta Gene Regul Mech 2020 08 8;1863(8):194578. Epub 2020 May 8.

Stem Cell Engineering, Center for Molecular and Cellular Bioengineering, Biotechnology Center, Technische Universität Dresden, Tatzberg 47, 01307 Dresden, Germany. Electronic address:

The Trithorax-related Set1 H3K4 methyltransferases are conserved from yeast to human. In yeast loss of Set1 causes pleiotropic effects but is compatible with life. In contrast, both mammalian Set1 orthologs: SETD1A and SETD1B are essential for embryonic development, however they have distinct functions. SETD1A is required shortly after epiblast formation whereas SETD1B becomes indispensible during early organogenesis. In adult mice both SETD1A and SETD1B regulate hematopoiesis differently: SETD1A is required for the establishment of definitive hematopoiesis whereas SETD1B is important for the maintenance of long-term hematopoietic stem cells. Both are implicated in different diseases with accumulating evidence for the association of SETD1A variants in neurological disorders and SETD1B variants with cancer. Why the two paralogs cannot or only partially compensate for the loss of each other is part of the puzzle that we try to sort out in this review.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194578DOI Listing
August 2020

Genes Polymorphism Depicts Developmental Disruption of Common Sole Eggs.

Open Life Sci 2019 Jan 31;14:549-563. Epub 2019 Dec 31.

Fytokou str, 38446, Volos, Greece.

In sole aquaculture production, consistency in the quality of produced eggs throughout the year is unpredictable. genes have a crucial role in controlling embryonic development and their genetic variation could alter the phenotype dramatically. In teleosts genome duplication led paralog genes to become diverged. Direct association of polymorphism in , & of with egg viability indicates as a potential genetic marker. High Resolution Melt (HRM) analysis was carried out in 52 viable and 61 non-viable eggs collected at 54±6 hours post fertilization (hpf). Allelic and genotypic frequencies of polymorphism were analyzed and results illustrated a significantly increased risk for non-viability for minor alleles and their homozygous genotypes. Haplotype analysis demonstrated a significant recessive effect on the risk of non-viability, by increasing the odds of disrupting embryonic development up to three-fold. Phylogenetic analysis showed that the paralog genes and , are separated distinctly in two clades and presented a significant ω variation, revealing their diverged evolutionary rate.
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http://dx.doi.org/10.1515/biol-2019-0061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874752PMC
January 2019

Aldh1b1 expression defines progenitor cells in the adult pancreas and is required for Kras-induced pancreatic cancer.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20679-20688. Epub 2019 Sep 23.

Paul Langerhans Institute Dresden, Helmholtz Center Munich at the University Clinic Carl Gustav Carus of Technische Universität Dresden, German Research Center for Environmental Health, Helmholtz Zentrum München, D-85764 Neuherberg, Germany;

The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of completely abrogates tumor development in a mouse model of Kras-induced pancreatic cancer.
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http://dx.doi.org/10.1073/pnas.1901075116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789925PMC
October 2019

Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs.

Blood 2019 10;134(15):1214-1226

Regeneration in Hematopoiesis and Animal Models in Hematopoiesis, Institute for Immunology Medical Faculty, Technische Universität (TU) Dresden, Dresden, Germany.

A major limitation preventing in vivo modulation of hematopoietic stem cells (HSCs) is the incomplete understanding of the cellular and molecular support of the microenvironment in regulating HSC fate decisions. Consequently, murine HSCs cannot be generated, maintained, or expanded in culture over extended periods of time. A significantly improved understanding of the bone marrow niche environment and its molecular interactions with HSCs is pivotal to overcoming this challenge. We here prospectively isolated all major nonhematopoietic cellular niche components and cross-correlate them in detail with niche cells defined by lineage marking or tracing. Compiling an extensive database of soluble and membrane-bound ligand-receptor interactions, we developed a computational method to infer potential cell-to-cell interactions based on transcriptome data of sorter-purified niche cells and hematopoietic stem and progenitor cell subpopulations. Thus, we establish a compendium of the molecular communication between defined niche components and HSCs. Our analysis suggests an important role for cytokine antagonists in the regulation of HSC functions.
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http://dx.doi.org/10.1182/blood.2019000176DOI Listing
October 2019

Inducible IL-7 Hyperexpression Influences Lymphocyte Homeostasis and Function and Increases Allograft Rejection.

Front Immunol 2019 10;10:742. Epub 2019 Apr 10.

Preclinical Approaches to Stem Cell Therapy/Diabetes, DFG-Center for Regenerative Therapies Dresden Cluster of Excellence, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220 c-kit Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8 T cells in secondary lymphoid organs, and reduced the proportion of CD4Foxp3 T regulatory cells. Of relevance to disease, conventional CD4 T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression and in a model of autoimmune diabetes . These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4 and CD8 T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.
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http://dx.doi.org/10.3389/fimmu.2019.00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467976PMC
September 2020

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

Development 2018 11 30;145(23). Epub 2018 Nov 30.

Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.
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http://dx.doi.org/10.1242/dev.169102DOI Listing
November 2018

Distinct pathways affected by menin versus MLL1/MLL2 in MLL-rearranged acute myeloid leukemia.

Exp Hematol 2019 01 10;69:37-42. Epub 2018 Oct 10.

Department of Pediatrics, University of Colorado, Denver/Anschutz Medical Campus. Aurora, CO, USA; Department of Pharmacology, University of Colorado, Denver/Anschutz Medical Campus. Aurora, CO, USA. Electronic address:

Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.
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http://dx.doi.org/10.1016/j.exphem.2018.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472707PMC
January 2019

A single reporter mouse line for Vika, Flp, Dre, and Cre-recombination.

Sci Rep 2018 09 27;8(1):14453. Epub 2018 Sep 27.

Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, 01307, Dresden, Germany.

Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.
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http://dx.doi.org/10.1038/s41598-018-32802-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160450PMC
September 2018

Human axial progenitors generate trunk neural crest cells in vitro.

Elife 2018 08 10;7. Epub 2018 Aug 10.

Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, United Kingdom.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.
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http://dx.doi.org/10.7554/eLife.35786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101942PMC
August 2018

Clonal Analysis Delineates Transcriptional Programs of Osteogenic and Adipogenic Lineages of Adult Mouse Skeletal Progenitors.

Stem Cell Reports 2018 07 21;11(1):212-227. Epub 2018 Jun 21.

Stem Cell Engineering, BIOTEC, Technische Universität Dresden, 01307 Dresden, Germany. Electronic address:

Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.
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http://dx.doi.org/10.1016/j.stemcr.2018.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067065PMC
July 2018

The H3K4 methyltransferase Setd1b is essential for hematopoietic stem and progenitor cell homeostasis in mice.

Elife 2018 06 19;7. Epub 2018 Jun 19.

Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, Dresden, Germany.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including , and . Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.
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http://dx.doi.org/10.7554/eLife.27157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025962PMC
June 2018

Inductive and Selective Effects of GSK3 and MEK Inhibition on Nanog Heterogeneity in Embryonic Stem Cells.

Stem Cell Reports 2018 07 17;11(1):58-69. Epub 2018 May 17.

Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland; Research Unit Stem Cell Dynamics, Helmholtz Zentrum München, 85764 Neuherberg, Germany. Electronic address:

Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.
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http://dx.doi.org/10.1016/j.stemcr.2018.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066909PMC
July 2018

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

Stem Cell Reports 2018 06 17;10(6):1895-1907. Epub 2018 May 17.

Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state.
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http://dx.doi.org/10.1016/j.stemcr.2018.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993559PMC
June 2018

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

PLoS One 2018 28;13(2):e0192437. Epub 2018 Feb 28.

Department of Pediatrics, University Clinic 'Carl Gustav Carus' Dresden, Dresden, Germany.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830289PMC
April 2018

lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1.

Cell Rep 2017 Dec;21(11):3012-3021

Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; German Cancer Research Center (DKFZ), Heidelberg and German Cancer Consortium (DKTK) partner site Dresden, 01307 Dresden, Germany; National Center for Tumor Diseases (NCT), University Hospital Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany. Electronic address:

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states.
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http://dx.doi.org/10.1016/j.celrep.2017.11.045DOI Listing
December 2017

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

Development 2017 12;144(23):4313-4321

Cellular Machines, Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, Dresden 01307, Germany

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.
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http://dx.doi.org/10.1242/dev.155218DOI Listing
December 2017

A COUP-TFII Human Embryonic Stem Cell Reporter Line to Identify and Select Atrial Cardiomyocytes.

Stem Cell Reports 2017 12 22;9(6):1765-1779. Epub 2017 Nov 22.

Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands; Department of Applied Stem Cell Technologies, MIRA Institute, University of Twente, Twente, the Netherlands. Electronic address:

Reporter cell lines have already proven valuable in identifying, tracking, and purifying cardiac subtypes and progenitors during differentiation of human pluripotent stem cells (hPSCs). We previously showed that chick ovalbumin upstream promoter transcription factor II (COUP-TFII) is highly enriched in human atrial cardiomyocytes (CMs), but not ventricular. Here, we targeted mCherry to the COUP-TFII genomic locus in hPSCs expressing GFP from the NKX2.5 locus. This dual atrial NKX2.5-COUP-TFII reporter line allowed identification and selection of GFP (G)/mCherry (M) CMs following cardiac differentiation. These cells exhibited transcriptional and functional properties of atrial CMs, whereas G/M CMs displayed ventricular characteristics. Via CRISPR/Cas9-mediated knockout, we demonstrated that COUP-TFII is not required for atrial specification in hPSCs. This new tool allowed selection of human atrial and ventricular CMs from mixed populations, of relevance for studying cardiac specification, developing human atrial disease models, and examining distinct effects of drugs on the atrium versus ventricle.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785710PMC
December 2017

Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass.

Nat Commun 2017 11 3;8(1):1297. Epub 2017 Nov 3.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, UPMC Paris-Sorbonne, 26 Rue d'Ulm, 75005, Paris, France.

X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks.
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http://dx.doi.org/10.1038/s41467-017-01415-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670228PMC
November 2017

, encoding a histone 3 lysine 4 methyltransferase, is a maternal effect gene required for the oogenic gene expression program.

Development 2017 07 15;144(14):2606-2617. Epub 2017 Jun 15.

Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany

Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific conditional knockout ( cKO) ovaries develop through all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that serves as maternal effect gene through regulation of the oocyte gene expression program.
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http://dx.doi.org/10.1242/dev.143347DOI Listing
July 2017

MLL2, Not MLL1, Plays a Major Role in Sustaining MLL-Rearranged Acute Myeloid Leukemia.

Cancer Cell 2017 06;31(6):755-770.e6

Department of Pediatrics, Section of Hematology/Oncology/BMT, University of Colorado, Denver/Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pharmacology, University of Colorado, Denver/Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

The MLL1 histone methyltransferase gene undergoes many distinct chromosomal rearrangements to yield poor-prognosis leukemia. The remaining wild-type allele is most commonly, but not always, retained. To what extent the wild-type allele contributes to leukemogenesis is unclear. Here we show, using rigorous, independent animal models, that endogenous MLL1 is dispensable for MLL-rearranged leukemia. Potential redundancy was addressed by co-deleting the closest paralog, Mll2. Surprisingly, Mll2 deletion alone had a significant impact on survival of MLL-AF9-transformed cells, and additional Mll1 loss further reduced viability and proliferation. We show that MLL1/MLL2 collaboration is not through redundancy, but regulation of distinct pathways. These findings highlight the relevance of MLL2 as a drug target in MLL-rearranged leukemia and suggest its broader significance in AML.
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http://dx.doi.org/10.1016/j.ccell.2017.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5598468PMC
June 2017

The contribution of homology arms to nuclease-assisted genome engineering.

Nucleic Acids Res 2017 Jul;45(13):8105-8115

Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.

Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine ∼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.
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http://dx.doi.org/10.1093/nar/gkx497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570031PMC
July 2017

TDRD6 mediates early steps of spliceosome maturation in primary spermatocytes.

PLoS Genet 2017 03 6;13(3):e1006660. Epub 2017 Mar 6.

Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes.
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http://dx.doi.org/10.1371/journal.pgen.1006660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358835PMC
March 2017

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.

Sci Rep 2016 05 24;6:25529. Epub 2016 May 24.

Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.

A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented.
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http://dx.doi.org/10.1038/srep25529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877586PMC
May 2016

Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis.

Stem Cell Reports 2016 Apr 31;6(4):525-538. Epub 2016 Mar 31.

German Center for Neurodegenerative Diseases (DZNE) Dresden, 01307 Dresden, Germany; CRTD - Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany. Electronic address:

The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.
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http://dx.doi.org/10.1016/j.stemcr.2016.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834051PMC
April 2016

Thy-1/β3 Integrin Interaction-Induced Apoptosis of Dermal Fibroblasts Is Mediated by Up-Regulation of FasL Expression.

J Invest Dermatol 2016 Feb 18;136(2):526-529. Epub 2015 Nov 18.

Department of Dermatology, Venereology and Allergology of Medical Faculty of Leipzig University, Leipzig, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2015.10.063DOI Listing
February 2016