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Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation

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Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation International Journal of Molecular Sciences Article Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation Indrek Teino 1,* , Antti Matvere 1 , Martin Pook 1, Inge Varik 1, Laura Pajusaar 1, 1 1 1 1 2,3, Keyt Uudeküll , Helen Vaher , Annika Trei , Arnold Kristjuhan ,Tõnis Org y 1, and Toivo Maimets y 1 Chair of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; [email protected] (A.M.); [email protected] (M.P.); [email protected] (I.V.); [email protected] (L.P.); [email protected] (K.U.); [email protected] (H.V.); [email protected] (A.T.); [email protected] (A.K.); [email protected] (T.M.) 2 Chair of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; [email protected] 3 Institute of Genomics, University of Tartu, Riia 23b, 51010 Tartu, Estonia * Correspondence: [email protected]; Tel.: +372-737-5023 These authors contributed equally. y Received: 6 November 2020; Accepted: 26 November 2020; Published: 28 November 2020 Abstract: Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which mediates the effects of a variety of environmental stimuli in multiple tissues. Recent advances in AHR biology have underlined its importance in cells with high developmental potency, including pluripotent stem cells. Nonetheless, there is little data on AHR expression and its role during the initial stages of stem cell differentiation. The purpose of this study was to investigate the temporal pattern of AHR expression during directed differentiation of human embryonic stem cells (hESC) into neural progenitor, early mesoderm and definitive endoderm cells. Additionally, we investigated the effect of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the gene expression profile in hESCs and differentiated cells by RNA-seq, accompanied by identification of AHR binding sites by ChIP-seq and epigenetic landscape analysis by ATAC-seq. We showed that AHR is differentially regulated in distinct lineages. We provided evidence that TCDD alters gene expression patterns in hESCs and during early differentiation. Additionally, we identified novel potential AHR target genes, which expand our understanding on the role of this protein in different cell types. Keywords: aryl hydrocarbon receptor; human embryonic stem cells; neural progenitors; early mesoderm; definitive endoderm; TCDD; RNA-seq; ChIP-seq; ATAC-seq 1. Introduction Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor belonging to the bHLH/PAS family of proteins. The unliganded AHR resides in the cytoplasm in complex with HSP90, AIP and p23. Upon activation by an agonist, AHR translocates to the nucleus where it is released from its cytoplasmic chaperones and dimerises with its nuclear partner ARNT (AHR nuclear translocator) [1,2]. This heterodimeric complex recognises and binds response elements in regulatory regions of its target genes, recruits cofactors and thereby modulates gene expression. Initially, AHR was identified as the mediator of toxic effects of various environmental contaminants, like polycyclic and halogenated aromatic hydrocarbons, including the most potent agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [3]. Activation of AHR by these chemicals leads to induction of various xenobiotic metabolising enzymes such as CYP1A1 and CYP1B1, which are responsible for the degradation and elimination of Int. J. Mol. Sci. 2020, 21, 9052; doi:10.3390/ijms21239052 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9052 2 of 24 these compounds from the cells. Later studies, however, have established that AHR can be activated by various endogenous and natural ligands, ascribing it an important role in cellular homeostasis, including regulation of the cell cycle, apoptosis, etc. The significance of AHR has been underscored, among others, in reproduction, liver homeostasis and the immune system [4–6]. In addition, recent advances in the field have emphasised the role of AHR in cells with high developmental potential i.e., stem cells. Several studies have investigated the role of AHR in adult stem cells using known antagonists or agonists such as TCDD. Hematopoietic stem cells (HSC), which descend from the mesoderm, have high therapeutic value but are limited in expansion capacities in vitro. Advances in this field, however, have shown that the AHR antagonist StemRegenin1 promotes expansion of HSC, retaining their developmental potential [7]. In addition, the RNA-binding protein Musashi-2 (MSI2) was shown to induce HSC self-renewal [8]. It was later established that MSI-2 exerts its effect through silencing of AHR signalling, with reduction of CYP1B1 expression being the key mechanism [9]. In addition, differentiation of mouse embryonic stem cells into cardiomyocytes has been shown to be interfered with by TCDD [10,11]. Accordingly, it was recently established that TCDD also disrupts differentiation of human embryonic stem cells (hESCs) into cardiomyocytes [12]. Interestingly, it was shown that the most profound effects appeared when TCDD-treatment was performed on hESCs rather than later during differentiation. Studies investigating the role of AHR in neural differentiation have revealed that activation of Ahr by TCDD suppresses neural precursor cell proliferation in a mouse model [13]. The mechanism included impaired G1/S cell cycle transition, downregulation of cyclin D1 and upregulation of p27 (CDKN1B) protein levels. The impact of TCDD on neuronal differentiation has been further elucidated during differentiation of human embryonic stem cells. It was shown that temporal exposure to TCDD increases neural rosette formation and the number of cells positive for MAP2 and TH, the latter being a key enzyme in dopamine synthesis. Additional studies have investigated AHR during endodermal differentiation. TCDD was found to impair hESC differentiation into the pancreatic lineage and alter DNA methylation [14]. Importantly, hypermethylation of PRKAG1, a regulator of insulin secretion, was observed. Embryonic stem cells (ESCs) are derived from the inner cell mass of a blastocyst. They are pluripotent, meaning that they can differentiate into three primordial lineages—ectoderm, endoderm and mesoderm—and give rise to every cell type of the body. Pluripotency is governed by specific transcription factors centred on OCT4, SOX2 and NANOG that enhance their own expression and suppress genes essential for differentiation. During differentiation of ESCs, pluripotency genes are generally downregulated, enabling upregulation of lineage-specific genes and thus lineage-specification. Differentiation of ESCs can be induced chemically, using small molecule compounds, as well as with growth factors, being also used in various combinations in commercially available differentiation media. In addition, formation of embryoid bodies (EBs), 3D structures that mimic in vivo differentiation, has been applied. Previous publications investigating AHR during the first steps of development have established that mouse Ahr is expressed at the 1-cell stage, downregulated at 2- and 8-cell stages followed by upregulation at later stages [15,16]. In mouse embryonic stem cells (mESCs), Ahr is repressed and upregulated during differentiation into EBs [17]. Importantly, Oct4, Sox2 and Nanog were shown to bind the distal promoter of Ahr, suppressing its expression and thereby promoting mESC mitotic progression and maintenance of pluripotency [18]. In human, AHR is expressed from the 1-cell stage throughout the blastocyst stage. AHR expression has been detected in various human embryonic stem cell (hESC) lines [19]. Importantly, AHR was reported to maintain the pluripotent state of hESCs and induce proliferation via kynurenine, an endogenous ligand of AHR, generated from tryptophan by IDO1 and TDO2 [19,20]. Consistent with this, AHR expression was downregulated following induction of ectodermal differentiation, ascribing AHR an important role in pluripotent hESCs as well as during differentiation [19]. There is little data about the temporal pattern of AHR during hESC differentiation. Considering this, our aims were to characterise AHR expression during induced differentiation of hESCs into neural Int. J. Mol. Sci. 2020, 21, 9052 3 of 24 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 25 progenitor,impact of TCDD definitive on the endoderm transcriptome and early of hESCs mesoderm as well cells. as Additionally,early differentiation we evaluated events theby RNA impact-seq, of TCDDcoupled on with the transcriptome identification of of hESCs genome as well-wide as earlyAHR di ffbindingerentiation sites events by ChIP by RNA-seq,-seq and coupled analysis with of identificationchromatin accessibility of genome-wide by ATAC AHR-seq. binding We expand sites by the ChIP-seq knowledge and on analysis the role of of chromatin AHR in stem accessibility cells by byidentifying ATAC-seq. its Wenovel expand potential the knowledgetarget genes on by the ChIP role-seq of AHRcoupled in stem with cells changes by identifying in gene expression its novel potentialdetermined target by RNA genes-seq. by ChIP-seq coupled with changes in gene expression determined by RNA-seq. 2.2. Results 2.1.2.1. AHR Is Downregulated during Non-DirectedNon-Directed DiDifferentiationfferentiation intointo EmbryoidEmbryoid BodiesBodies (EBs)(EBs) ToTo gaingain first first insight insight into into the regulationthe regulation of AHR of duringAHR earlyduring di ffearlyerentiation, differentiation,
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