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© The Author(s) 2020. ; 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 protein 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. ; This work was supported by grants to P.M.F-S. from the Spanish Ministry of Economy and Competitiveness (SAF2014-51813-R and SAF2017-82597-R) and from the Junta de Extremadura (GR15008 and IB160210). Research at P.M.F-S. laboratory was also funded by the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). A.C.R. was funded by a long-term FEBS Fellowship. L.M. work was funded by MINECO Grants BIO2012-39980 and BIO2015-70978-R. All Spanish funding is co-sponsored by the European Union FEDER program.

Non-small cell lung adenocarcinoma (NSCLC) bearing K-RasG12D mutations is one of the most prevalent types of lung cancer worldwide. Aryl hydrocarbon receptor (AHR) expression varies in human lung tumors and has been associated with either increased or reduced lung metastasis. In the mouse, Ahr also adjusts lung regeneration upon injury by limiting the expansion of resident stem cells. Here, we show that the loss of Ahr enhances K-RasG12D-driven NSCLC in mice through the amplification of stem cell subpopulations. Consistent with this, we show that K-RasG12D;Ahr−/− lungs contain larger numbers of cells expressing markers for both progenitor Clara (SCGB1A1 and CC10) and alveolar type-II (SFTPC) cells when compared to K-RasG12D;Ahr+/+-driven tumors. They also have elevated numbers of cells positive for pluripotent stem cells markers such as SOX2, ALDH1, EPCAM, LGR5 and PORCN. Typical pluripotency genes Nanog, Sox2 and c-Myc were also upregulated in K-RasG12D;Ahr−/− lung tumors as found by RNAseq analysis. In line with this, purified K-RasG12D/+;Ahr−/− lung cells generate larger numbers of organoids in culture that can subsequently differentiate into bronchioalveolar structures enriched in both pluripotency and stemness genes. Collectively, these data indicate that Ahr antagonizes K-RasG12D-driven NSCLC by restricting the number of cancer-initiating stem cells. They also suggest that Ahr expression might represent a good prognostic marker to determine the progression of K-RasG12D-positive NSCLC patients. ; This work was supported by grants to P.M.F.-S. from the Spanish Ministry of Economy and Competitiveness (SAF2017-82597-R and PID2020-114846RB-I00) and from the Junta de Extremadura (GR18006, IB160210 and IB20014). A.N.-P. was supported by the Ministry of Economy and Competitiveness and the Junta de Extremadura. C.M.R.-G. was a predoctoral F.P.U. fellow of the Ministry of Economy and Competitiveness. All Spanish funding was co-sponsored by the European Union FEDER program.

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions. ; Ministerio de Economía y Competitividad [BFU2011-22678, SAF2014-51813-R to P.M.F-S.]; Junta de Extremadura [GR10008, GR15008]; Red Temática de Investigación Cooperativa en Cáncer (RTICC); Carlos III Institute; Spanish Ministry of Economy and Competitiveness [RD12/0036/0032]; FPI Fellowship from the Junta de Extremadura (to A.M.H.); Marie Curie IRG project (FP7-PEOPLE-2007-4-3-IRG: SOMATIC LINE-1, in part to A.M.); CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489, FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II (to J.L.G.P.); European Research Council [ERC-Consolidator ERC-STG-2012-233764]; International Early Career Scientist grant from the Howard Hughes Medical Institute [IECS-55007420]; European Union FEDER program. Funding for open access charge: Ministerio de Economía y Competitividad [BFU2011-22678, SAF2014-51813-R to P.M.F-S.].

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR and AhR mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR than in AhR testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Une pectedly, AhR males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR2/2 ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR\+/\+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements. ; This work was supported by grants to P.M.F-S. from the Spanish Ministry of Science and Innovation (BFU2011-22678) and Economy and Competitiveness (SAF2014-51813-R) and from the Junta de Extremadura (GR15008). Research at the F.J.P. laboratory was supported by grant no. AGL2013-43211-R from the Spanish Ministry of Economy and Competitiveness. Research at the P.M.F-S laboratory was also supported by the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Carlos III Institute, Spanish Ministry of Economy and Competitiveness (RD12/0036/0032). E.R.L., F.J.G-R and E.B. were supported by the RTICC, MICINN and Junta de Extremadura, respectively. N.M.M. was an FPI fellow of the Spanish Ministry of Education. P.M.M. was supported by a pre-doctoral grant from the Spanish Ministry of Education, Culture and Sport (FPU13/03991) C.O.F. was supported by a postdoctoral Juan de la Cierva (IJCI-2014-21671) grant from the Spanish Ministry of Economy and Competitiveness. A.M.P. and E.L. were supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-59307-R), MEIONet and the Junta de Castilla y Leon. All Spanish funding is co-sponsored by the European Union FEDER programme. ; Peer Reviewed