Blog
About

0
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and Is Resilient to TGFβ1 Activation

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%–70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression.

          Related collections

          Most cited references 47

          • Record: found
          • Abstract: found
          • Article: not found

          Functional annotation of native enhancers with a Cas9 -histone demethylase fusion

          Understanding of mammalian enhancer function is limited by the lack of a technology to rapidly and thoroughly test their cell type-specific function. Here, we use a nuclease-deficient (d)Cas9 histone demethylase fusion to functionally characterize previously described and novel enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Dynamics and memory of heterochromatin in living cells.

            Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome. Copyright © 2012 Elsevier Inc. All rights reserved.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Editing the epigenome: technologies for programmable transcription and epigenetic modulation.

              Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.
                Bookmark

                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                21 May 2020
                May 2020
                : 21
                : 10
                Affiliations
                [1 ]Epigenetic Editing Laboratory, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1 EA11, 9713 GZ Groningen, The Netherlands; gjaltema@ 123456molgen.mpg.de (R.A.F.G.); d.goubert@ 123456umcg.nl (D.G.); kriztian.huisman@ 123456gmail.com (C.H.); conpily@ 123456gmail.com (C.d.P.G.T.); p.g.jellema@ 123456umcg.nl (P.G.J.); wwwgrass123@ 123456163.com (D.W.); u.brouwer@ 123456umcg.nl (U.B.)
                [2 ]MATRIX Research Group, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands; r.a.bank@ 123456umcg.nl
                [3 ]Institute of Biochemistry, Biological Research Centre, H-6726 Szeged, Hungary; konczmisa@ 123456gmail.com (M.K.); kiss.antal@ 123456brc.hu (A.K.)
                [4 ]Doctoral School of Biology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Hungary
                [5 ]Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands; P.J.Verschure@ 123456uva.nl
                Author notes
                [* ]Correspondence: m.g.rots@ 123456umcg.nl ; Tel.: +31-50-3610153
                [†]

                Both Authors Contributed Equally.

                Article
                ijms-21-03634
                10.3390/ijms21103634
                7279273
                32455614
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                Categories
                Article

                Molecular biology

                epigenetic editing, krab, gene repression, plod2, fibrosis, cancer

                Comments

                Comment on this article