Alu retrotransposons modulate Nanog expression through dynamic changes in regional chromatin conformation via aryl hydrocarbon receptor

González‑Rico et al. Epigenetics & Chromatin https://doi.org/10.1186/s13072-020-00336-w (2020) 13:15 Epigenetics & Chromatin Open Access RESEARCH Alu retrotransposons modulate Nanog expression through dynamic changes in regional chromatin conformation via aryl hydrocarbon receptor Francisco J. González‑Rico1, Cristina Vicente‑García2,3, Almudena Fernández2,3, Diego Muñoz‑Santos2,3, Lluís Montoliu2,3, Antonio Morales‑Hernández1, Jaime M. Merino1, Angel‑Carlos Román1* and Pedro M. Fernández‑Salguero1* Abstract Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator pro‑ tein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation. Keywords: Alu retrotransposons, Aryl hydrocarbon receptor, Differentiation, Nanog, Chromatin conformation Introduction Cellular differentiation is a key process during embryonic development [1, 2] and in adult stem cell homeostasis [3] whose alteration can lead to pathological states including cancer [4, 5]. In the last few years, transcriptional regulatory mechanisms that control pluripotency and differentiation have been described (see review in [6]). Taking advantage of this knowledge, specific techniques have been recently developed to revert differentiated *Correspondence: ; 1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Av. de Elvas s/n, 06071 Badajoz, Spain Full list of author information is available at the end of the article cells to an induced pluripotent stem cell phenotype [7, 8]. Some of these transcription factors are considered master regulators of pluripotency and include Nanog, Oct4, Sox2, c-Myc and KLF4, among others [6]. Nanog was first described as an embryo-specific homeobox gene [9]. Later on, two independent groups found that it was required for the maintenance of pluripotency in embryonic stem cells [10, 11], in which it acts as transcriptional activator of genes related to pluripotency and as transcriptional repressor of genes involved in differentiation [12]. Nanog expression can be self-induced [12] in embryonic stem cells or activated by different transcription factors like Oct4 and Sox2 [13] or FoxD3 [14]. Upon differentiation, proteins such as p53, Foxa1, RIP140 © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. 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 in a credit line to the data. González‑Rico et al. Epigenetics & Chromatin (2020) 13:15 or the aryl hydrocarbon receptor (AhR) are also able to repress Nanog [15–18]. These changes in Nanog expression and in the levels of other pluripotency regulators involve epigenetic modifications of their loci, as observed in several differentiation models [18–23]. Under stem cell differentiating conditions, chromatin folding is altered, the chromatin of topologically associated domains (TADs) is reordered and the whole transcriptome of the cell rewired [24–28]. Nevertheless, we are still far from a comprehensive view of the molecular events that comprise cellular differentiation and on the functions of specific regulators of pluripotency. We recently found that the transcription factor AhR was required for retinoic acid (RA)-mediated differentiation of NTERA-2 cells (hereafter NTERA) [17]. Specifically, RA-induced differentiation promoted AhR binding to Alu retrotransposons flanking pluripotency genes Nanog and Oct4. Notably, Alu-generated transcripts in differentiated cells were able to repress Nanog and Oct4 expression by a mechanism involving the miRNA machinery [17]. In addition, these results are in agreement with the proposed roles for repetitive elements as enhancer–promoter insulators and/or chromatin barriers and architecture [29–34]. In this work, we have investigated if Alu retrotransposons located near pluripotency genes could participate in human stem cell differentiation by modulating chromatin structure and dynamics. To test such possibility, we focused on the changes in chromatin folding surrounding the Nanog locus that could take place during NTERA differentiation. Our main conclusion is that a molecular complex, composed by AhR, PRMT1, CHAF1B and CTCF, interacts with Alu elements modifying the epigenetic profile and generating a chromatin loop around the Nanog gene that will lead to its repression during RA-mediated differentiation. In fact, impairing the interaction between AhR and PRMT1 with the Alu elements restored Nanog expression in differentiation-induced cells. Results Alu elements located flanking the Nanog locus have enhancer‑blocking activity We have recently found that transcriptional downregulation of Nanog during NTERA cell differentiation was dependent on the upregulation of AhR and on its binding to repetitive sequences neighboring the Nanog locus, being those 7SL RNA-derived human retroelements (Alu family) [17]. Following the same bioinformatic algorithm used to analyze the mouse heterologous of these human repetitive elements (e.g., B1 family) [35], we have extracted those Alu retro (...truncated)