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      Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene

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          Abstract

          The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/ Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus.

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          Most cited references37

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          The cis-Regulatory Atlas of the Mouse Immune System

          A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell-types that span the mouse immune system and its differentiation cascades. This breadth of data enables variance components analysis which suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by ChIP-seq. Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation, and a foundational scaffold to define key regulatory events throughout the immunological genome. A cis-regulatory map of the mouse immune system illuminates gene expression patterns and regulatory logic across 86 primary cell types and pairs immune transcription factors with cell type-specific regulatory elements.
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            Noncoding transcription at enhancers: general principles and functional models.

            Mammalian genomes are extensively transcribed outside the borders of protein-coding genes. Genome-wide studies recently demonstrated that cis-regulatory genomic elements implicated in transcriptional control, such as enhancers and locus-control regions, represent major sites of extragenic noncoding transcription. Enhancer-templated transcripts provide a quantitatively small contribution to the total amount of cellular nonribosomal RNA; nevertheless, the possibility that enhancer transcription and the resulting enhancer RNAs may, in some cases, have functional roles, rather than represent mere transcriptional noise at accessible genomic regions, is supported by an increasing amount of experimental data. In this article we review the current knowledge on enhancer transcription and its functional implications.
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              Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters.

              Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell-specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4-10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome.
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                Author and article information

                Contributors
                Role: Writing – original draft
                Role: MethodologyRole: Validation
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Supervision
                Role: Methodology
                Role: Methodology
                Role: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                26 May 2020
                2020
                : 15
                : 5
                : e0233191
                Affiliations
                [1 ] Aix-Marseille University, Inserm, TAGC, UMR1090, Marseille, France
                [2 ] Equipe Labélisée Ligue Contre le Cancer, Marseille, France
                [3 ] Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
                [4 ] CNRS UMR7104, Illkirch, France
                [5 ] INSERM U1258, Illkirch, France
                [6 ] University of Strasbourg, Illkirch, France
                [7 ] Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Montpellier, France
                University of Crete & IMBB-FORTH, GREECE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist

                [¤]

                Current address: Vinmec Research Institute of Stem cell and Gene technology (VRISG), Hà Nội, Vietnam

                Author information
                http://orcid.org/0000-0002-8101-7108
                Article
                PONE-D-20-02571
                10.1371/journal.pone.0233191
                7250416
                32453736
                6cb54d60-e63f-4164-8e2d-6d38865f03d9
                © 2020 Alomairi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 January 2020
                : 29 April 2020
                Page count
                Figures: 4, Tables: 0, Pages: 16
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100007586, Aix-Marseille Université;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004099, Ligue Contre le Cancer;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006331, Canceropôle PACA;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001665, Agence Nationale de la Recherche;
                Award ID: ANR-11-IDEX-0001-02
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001665, Agence Nationale de la Recherche;
                Award ID: ANR-10-LABX-0030-INRT
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100010663, H2020 European Research Council;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001665, Agence Nationale de la Recherche;
                Award ID: ANR-10-IDEX-0002-02
                Award Recipient :
                We thank the Transcriptomics and Genomics Marseille-Luminy (TGML) platform for sequencing the ChIP-seq samples and the Marseille-Luminy cell biology platform for the management of cell culture. Sequencing of 4C samples was performed by the IGBMC GenomEast platform. TGML and GenomEast platforms are member of the France Genomique consortium (ANR-10-INBS-0009). Work in the laboratory of S.S. was supported by recurrent funding from INSERM and Aix-Marseille University and specific grants from the Ligue contre le Cancer (Equipe Labellisée LIGUE 2018), the Agence Nationale pour la Recherche, ANR (ANR-17-CE12-0035; ANR-18-CE12-0019), Cancéropôle PACA, Institut National contre le Cancer (PLBIO018-031 INCA_12619), the Excellence Initiative of Aix-Marseille University - A*Midex, a French “Investissements d’Avenir” programme (ANR-11-IDEX-0001-02). Work in the lab of TS is supported by funds from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Starting Grant 678624 - CHROMTOPOLOGY), the ATIP-Avenir program, and the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02. AMM is supported by funds from IDEX (University of Strasbourg) and the Institut National du Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Hematopoietic Progenitor Cells
                Thymocytes
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Genomic Signal Processing
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromatin
                Biology and Life Sciences
                Genetics
                Epigenetics
                Chromatin
                Biology and Life Sciences
                Genetics
                Gene Expression
                Chromatin
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Enhancer Elements
                Custom metadata
                ChIP-seq and 4C-seq data described in this study are available in GEO database under the accession number GSE147234 ( http://www.ncbi.nlm.nih.gov/geo/).

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