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      Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition

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          Abstract

          The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser 224-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser 224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser 224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.

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          A map of the cis-regulatory sequences in the mouse genome.

          The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 10(9) bases of the mouse genome possess a high degree of conservation with the human genome, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized into domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.
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            Design and analysis of ChIP-seq experiments for DNA-binding proteins

            Recent progress in massively parallel sequencing platforms has allowed for genome-wide measurements of DNA-associated proteins using a combination of chromatin immunoprecipitation and sequencing (ChIP-seq). While a variety of methods exist for analysis of the established microarray alternative (ChIP-chip), few approaches have been described for processing ChIP-seq data. To fill this gap, we propose an analysis pipeline specifically designed to detect protein binding positions with high accuracy. Using three separate datasets, we illustrate new methods for improving tag alignment and correcting for background signals. We also compare sensitivity and spatial precision of several novel and previously described binding detection algorithms. Finally, we analyze the relationship between the depth of sequencing and characteristics of the detected binding positions, and provide a method for estimating the sequencing depth necessary for a desired coverage of protein binding sites.
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              Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.

              Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                24 January 2019
                2019
                : 8
                : e42341
                Affiliations
                [1 ]deptDepartment of Molecular Biology Howard Hughes Medical Institute, Massachusetts General Hospital BostonUnited States
                [2 ]deptDepartment of Genetics Harvard Medical School BostonUnited States
                [3 ]deptKoch Institute for Integrative Cancer Research Massachusetts Institute of Technology CambridgeUnited States
                [4 ]deptDepartment of Molecular Biology Massachusetts General Hospital BostonUnited States
                [5 ]Cell Signaling Technology DanversUnited States
                Weill Cornell Medicine United States
                Weill Cornell Medicine United States
                Weill Cornell Medicine United States
                Temple University School of Medicine United States
                Author notes
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-9976-1541
                http://orcid.org/0000-0002-3395-4498
                http://orcid.org/0000-0001-7786-8850
                Article
                42341
                10.7554/eLife.42341
                6361588
                30676316
                df426970-01ce-4160-b93a-2335c59a8cf8
                © 2019, Del Rosario et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 25 September 2018
                : 23 January 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: NSF GRFP
                Award Recipient :
                Funded by: Herchel Smith Endowment fund;
                Award ID: Herchel Smith Graduate Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R37-GM58839
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Tools and Resources
                Chromosomes and Gene Expression
                Custom metadata
                The chromosome architectural protein, CTCF, is phosphorylated at Ser 224 in a cell-cycle-dependent manner and abrogation of phosphorylation leads to a cell growth defect.

                Life sciences
                ctcf,post-translational modification,cell cycle,chromosome architecture,plk1,mouse
                Life sciences
                ctcf, post-translational modification, cell cycle, chromosome architecture, plk1, mouse

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