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Depletion of DNMT1 in Differentiated Human Cells Highlights Key Classes of Sensitive Genes and an Interplay with Polycomb Repression

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Depletion of DNMT1 in Differentiated Human Cells Highlights Key Classes of Sensitive Genes and an Interplay with Polycomb Repression Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression O'Neill, K. M., Irwin, R. E., Mackin, S-J., Thursby, S-J., Thakur, A., Bertens, C., Masala, L., Loughery, J. E. P., McArt, D. G., & Walsh, C. P. (2018). Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression. Epigenetics & chromatin, 11, [12]. https://doi.org/10.1186/s13072-018-0182-4 Published in: Epigenetics & chromatin Document Version: Publisher's PDF, also known as Version of record Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights Copyright 2018 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact [email protected]. Download date:07. Oct. 2021 O’Neill et al. Epigenetics & Chromatin (2018) 11:12 https://doi.org/10.1186/s13072-018-0182-4 Epigenetics & Chromatin RESEARCH Open Access Depletion of DNMT1 in diferentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression Karla M. O’Neill1,5†, Rachelle E. Irwin1†, Sarah‑Jayne Mackin1, Sara‑Jayne Thursby1, Avinash Thakur1,6, Ciske Bertens1,2, Laura Masala1,3, Jayne E. P. Loughery1, Darragh G. McArt4 and Colum P. Walsh1* Abstract Background: DNA methylation plays a vital role in the cell, but loss-of-function mutations of the maintenance meth‑ yltransferase DNMT1 in normal human cells are lethal, precluding target identifcation, and existing hypomorphic lines are tumour cells. We generated instead a hypomorphic series in normal hTERT-immortalised fbroblasts using stably integrated short hairpin RNA. Results: Approximately two-thirds of sites showed demethylation as expected, with one-third showing hypermeth‑ ylation, and targets were shared between the three independently derived lines. Enrichment analysis indicated sig‑ nifcant losses at promoters and gene bodies with four gene classes most afected: (1) protocadherins, which are key to neural cell identity; (2) genes involved in fat homoeostasis/body mass determination; (3) olfactory receptors and (4) cancer/testis antigen (CTA) genes. Overall efects on transcription were relatively small in these fbroblasts, but CTA genes showed robust derepression. Comparison with siRNA-treated cells indicated that shRNA lines show substantial remethylation over time. Regions showing persistent hypomethylation in the shRNA lines were associated with poly‑ comb repression and were derepressed on addition of an EZH2 inhibitor. Persistent hypermethylation in shRNA lines was, in contrast, associated with poised promoters. Conclusions: We have assessed for the frst time the efects of chronic depletion of DNMT1 in an untransformed, dif‑ ferentiated human cell type. Our results suggest polycomb marking blocks remethylation and indicate the sensitivity of key neural, adipose and cancer-associated genes to loss of maintenance methylation activity. Keywords: DNMT1, EZH2, Protocadherin, Body mass, Cancer/testis antigen Background 1 (DNMT1) enzyme [2]. Methylation is known to play DNA methylation is an important mechanism for epige- an important role in regulating imprinted loci [3], genes netic regulation of genes in both mouse and human [1]. It on the inactive X chromosome [4] and germline-specifc occurs mainly at the CpG dinucleotide, and methylation genes [5] in mouse. at this symmetrical site is efciently maintained during Where methylation occurs at the promoter of a gene, it replication by the action of the DNA methyltransferase is strongly associated with the silencing of transcription, particularly if there is a high density of CpGs, a so-called *Correspondence: [email protected] CpG island (CGI). However, studies have shown that †Karla M. O’Neill and Rachelle E. Irwin contributed equally to this work most CGI are intrinsically protected from methylation [6, 1 Genomic Medicine Research Group, Centre for Molecular Biosciences, 7] and only a small number shows dynamic changes dur- School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine BT52 1SA, UK ing development, mostly in the three classes mentioned Full list of author information is available at the end of the article above [5, 8], though there may be others which have not © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. O’Neill et al. Epigenetics & Chromatin (2018) 11:12 Page 2 of 21 yet been clearly defned. As you move outward from an knockout alleles. Acute depletion of DNMT1 using an island, the shores and shelves show higher levels of meth- siRNA-mediated approach in embryonal carcinoma cells ylation and greater dynamic response [9], though here the also found regions of low CpG density (open sea, shelf) to link to changes in gene activity is less clear [10]. Methyla- be the most afected by loss of methylation [70]. Among tion is also associated with larger regions of inert chro- the small number of dysregulated genes, there was some matin, such as the inactive X, pericentromeric repeats enrichment for cell morphogenesis and phosphorylation and regions rich in transposable elements [1], generally pathways. consistent with a repressive role. Recent genome-wide Neither of these cancer cell lines, however, are a good surveys have also indicated that high levels of methyla- model for the normal diferentiated cell as they are trans- tion are found in the bodies of active genes, where they formed, aneuploid, hypermethylated, and contain a may facilitate transcription [11, 12]. In keeping with this, number of diferent mutations in key regulatory genes. we and others recently showed that artifcially decreasing Additionally, acute depletion of DNMT1 results in cell intragenic methylation levels reduced steady-state tran- cycle delay, triggering of the DNA damage response and script levels, consistent with a positive role for methyla- increased rates of cell death [24, 25, 30], making it dif- tion in the gene body [11–13]. cult to separate acute and chronic efects. Another major system for epigenetic repression is via To circumvent some of the difculties outlined above, histone modifcation, particularly by the polycomb group we generated a series of isogenic human cell lines derived of proteins, with EZH2 being one of the main enzymes from the hTERT-immortalised normal fbroblast line involved [14]. A number of studies suggest an interplay hTERT1604 as previously described [30]. Tese are nor- between polycomb- and DNMT-mediated repression, mosomic and non-transformed, and by using a stably with a generally negative correlation between DNA meth- incorporated plasmid with an shRNA targeting DNMT1 ylation and the H3K27me3 mark deposited by EZH2 [15, we were able to isolate a number of clonally derived lines 16]. Supporting this, a loss of DNA methylation caused to allow identifcation of any cell line-specifc efects. a reshaping of the histone landscape and derepression of While these showed initially the range of shared features some polycomb targets in mouse ES cells [17], suggesting indicative of a global response to the loss of this criti- that DNA methylation helps to determine where poly- cal regulator, including cell cycle delay, demethylation comb marks are deposited. of imprinted genes and others, they could be cultured While DNMT1 is the main maintenance methyltrans- for longer under selection [30], allowing identifcation ferase, there also appears to be an important role for the of loci with particular sensitivity for decreased main- de novo enzymes DNMT3A and DNMT3B in comple- tenance methyltransferase activity. Here we set out to menting that activity at some loci [18, 19]. In order to completely characterise the methylation changes seen in clarify which genes are most sensitive to DNMT1 loss the cell lines using the Illumina Infnium HumanMeth- in human, a number of studies have been carried out ylation450 BeadChip (450k) array platform [31] and sub- using mutations within the gene to assess the efects of sequent analysis using the RnBeads pipeline [32]. Tese loss of methylation [19–22]. While this
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