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Hmga2 Is Necessary for Otx2-Dependent Exit of Embryonic

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Hmga2 Is Necessary for Otx2-Dependent Exit of Embryonic Navarra et al. BMC Biology (2016) 14:24 DOI 10.1186/s12915-016-0246-5 RESEARCH ARTICLE Open Access Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state Angelica Navarra1,2, Anna Musto1,2, Anna Gargiulo1,2, Giuseppe Petrosino3, Giovanna Maria Pierantoni1,4, Alfredo Fusco1,4, Tommaso Russo1,2* and Silvia Parisi1,2* Abstract Background: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the exit from the pluripotent ground state that leads to the acquisition of the ‘primed’ pluripotent phenotype, characteristic of the epiblast-like stem cells (EpiLCs). The transcription factors Oct4 and Otx2 play a key role in this phenomenon. In particular, Otx2 pioneers and activates new enhancers, which are silent in ESCs and which control the transcription of genes responsible for the acquisition of the EpiLC phenotype. An important point that remains to be addressed is the mechanism through which Otx2 engages the new enhancers and stably associates with them. Hmga2 is a member of the high-mobility group family of proteins, non-histone components of chromatin whose expression is high during embryogenesis and becomes low or undetectable in adults. Its high expression during embryogenesis suggests that Hmga2 fulfills important roles in development. Results: Here, we demonstrate that Hmga2 accumulates soon after the induction of ESC differentiation. Its suppression hampers the exit of ESCs from the pluripotent ground state and their differentiation into EpiLCs. Mechanistically, Hmga2 controls the differentiation process by cooperating with Otx2 in the pioneering of new enhancers. In Hmga2 null induced pluripotent stem cells we observe that Otx2 fails to regulate its target genes upon the induction of differentiation. Hmga2 associates to Otx2-bound loci in EpiLCs, and in Hmga2 KO cells Otx2 is unable to engage and activate the new enhancers, thus indicating that Hmga2 is required for the binding of Otx2 to its cis-elements. We find that this mechanism also operates on the Hmga2 gene, which is one of the targets of Otx2, thus indicating the existence of a positive feedback loop. Conclusions: Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state. Upon the induction of ESC differentiation, Otx2 alone or in combination with Oct4 engages new enhancers, which are silent in undifferentiated ESCs. The Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state. Keywords: Embryonic stem cell differentiation, Enhancer activation, High-mobility group protein * Correspondence: [email protected]; [email protected] 1Department of Molecular Medicine and Medical Biotechnology, University of Napoli Federico II, via S. Pansini 5, 80131 Naples, Italy Full list of author information is available at the end of the article © 2016 Navarra et al. Open Access 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. Navarra et al. BMC Biology (2016) 14:24 Page 2 of 17 Background the IFN-β gene depends on the recruitment of several Embryonic stem cells (ESCs) are able to self-renew and TFs, including NFkB, to an enhancer region positioned in differentiate in vitro, giving rise to all the cell types of the IFN-β gene promoter. The assembly of this complex is the embryo, thus mimicking the events that take place dependent on the interaction of Hmga1 with an A/T-rich in vivo during the early stages of development. While sequence present in the promoter [14]. Another well- enormous progress has been made in understanding the studied example of the role of Hmga in enhanceosome mechanisms that sustain ESC pluripotency [1], the pro- formation is that of the IL-2Rα gene. In this case, Hmga1 cesses governing the exit of ESCs from the pluripotent is upregulated upon stimulation of T cells and binds to A/ state and the early steps of differentiation are still not T-rich sequences in the IL-2Rα gene promoter. This re- well understood. It is well established that activation of cruitment induces a chromatin remodeling that allows the ERK and GSK3β pathways is necessary for lineage several TFs, like Elf-1, STAT5 and others, to interact with commitment of ESCs [2], and that MAPK phosphatases their cognate cis-elements, which become accessible only have an important role in the downregulation of ERK as a consequence of Hmga1-DNA interaction [15]. In the signaling [3]. Recent results indicate that redistribution case of the IL-2Rα gene promoter, and also in the case of of βHLH transcription factor (TF) Tfe3 from the nucleus the α-B-crystallin gene [16], Hmga proteins interact with to the cytoplasm, driven by folliculin/Fnip1 and Tsc2, is A/T-rich sequences positioned on the surface of, or close necessary for the exit from the pluripotent state [4]. An- to, positioned nucleosomes which hamper the binding of other βHLH TF, Tcf15, primes ESCs for differentiation sequence-specific TFs. One of the effects of Hmga seems by downregulating Nanog and inducing Otx2 [5]. Of par- to be that of removing the nucleosomal constraints that ticular interest is the role of Otx2. This TF, alone and in prevent the formation of the TF-DNA complexes [13]. combination with Oct4, regulates a complex array of Here, we show that Hmga2 accumulates upon the in- genes that marks the very early steps of exit from pluri- duction of mouse ESC differentiation and its suppression potency and the acquisition of early post-implantation interferes with the transition of ESCs into the epiblast- epiblast cell state (EpiLC) [6–8]. Induction of Otx2 ex- like state. Hmga2 gene transcription is regulated by pression and its engagement to genomic loci occur very Otx2 and accumulating Hmga2 cooperates with Otx2 in early during the exit of mouse ESCs from the ground the pioneering activity that allows the switching on of state of pluripotency, indicating that Otx2 acts as a pion- new enhancers that regulate change in the gene expres- eer to engage and activate silent enhancers. However, sion program that ultimately governs the exit from the while accumulation of Otx2 clearly contributes to its pluripotent ground state of ESCs. own activity, how Otx2 engages new enhancers and sta- bly associates with them remains unclear. Results The high-mobility group (HMG) family of proteins Hmga2 suppression interferes with ESC differentiation form an abundant, heterogeneous, non-histone compo- The screening of 20,850 shRNAs interfering with 11,250 nent of chromatin. Hmga members of this family are independent transcripts, aimed at searching for factors highly expressed during embryogenesis and their expres- involved in the differentiation of ESCs [17], allowed us sion becomes more restricted as fetal development pro- to identify, among numerous factors that hamper ESC gresses, with low or undetectable expression in adults [9, differentiation, the Hmga2 gene. Using an ESC line in 10], and becomes abundant in malignant cells in vitro which GFP is under the control of the Nanog gene pro- and in vivo, where they have been extensively studied moter, we found that, at 8 days after the induction of dif- [11]. The high expression of Hmga proteins during em- ferentiation, the number of undifferentiated cells, thus still bryogenesis suggests that they fulfill important roles in expressing GFP, was significantly higher in Hmga2 knock- development. Indeed, it has been recently reported that down (KD) cells than in control shRNA-transfected cells, the Hmga1/Hmga2 double knockout (KO) mice showed while the number of differentiated β3-tubulin positive high embryonic lethality [12]. cells was decreased (Fig. 1a). To measure the effects of Hmga proteins lack transcriptional activity per se, but Hmga2 suppression, we induced the differentiation of the act by orchestrating the assembly of transcription factor E14Tg2a ESC line through the formation of serum-free complexes also known as enhanceosomes [13]. They pref- embryoid bodies (SFEBs). As shown in Fig. 1b, wildtype erentially bind to A/T-rich sequences in close proximity (wt) SFEBs express differentiation-specific markers, like to, or overlapping with, the binding sites of sequence- the neural precursor marker Sox1, starting around day 2 specific TFs and favor the formation of multi-subunit of differentiation, while the inner part of the SFEBs con- protein-DNA complexes by modifying the chromatin tains small groups of cells that express stemness markers structure in an ATP-independent fashion [13]. One ex- like Oct4 and Nanog. The silencing of Hmga2 with siR- ample of the function of Hmga proteins is that of the IFN- NAs caused an evident change in the SFEB phenotype, β gene promoter. Upon viral infection, the transcription of which showed a decreased number of Sox1 positive cells Navarra et al. BMC Biology (2016) 14:24 Page 3 of 17 Fig. 1 (See legend on next page.) Navarra et al. BMC Biology (2016) 14:24 Page 4 of 17 (See figure on previous page.) Fig. 1 Hmga2 suppression interferes with embryonic stem cell (ESC) differentiation. a ESCs expressing GFP under the control of Nanog gene promoter were transfected with the control shRNA (sh Ctrl) and two different shRNAs targeting Hmga2, which induced a significant decrease of the Hmga2 protein.
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