The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells
2020; Elsevier BV; Volume: 295; Issue: 28 Linguagem: Inglês
10.1074/jbc.ra120.012753
ISSN1083-351X
AutoresHiromi Murata, Susumu Tanaka, Tomoko Tsuzuki‐Nakao, Takeharu Kido, Maiko Kakita‐Kobayashi, Naoko Kida, Yoji Hisamatsu, Hiroaki Tsubokura, Yoshiko Hashimoto, Masaaki Kitada, Hidetaka Okada,
Tópico(s)Endometriosis Research and Treatment
ResumoCyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart– and neural crest derivatives–expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs. Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart– and neural crest derivatives–expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs. The human endometrium undergoes growth and regeneration during each menstrual cycle. In the absence of an implantation, the endometrium sheds, is repaired, proliferates, and terminally differentiates to prepare for subsequent embryo implantation (1Cha J. Sun X. Dey S.K. Mechanisms of implantation: strategies for successful pregnancy.Nat. Med. 2012; 18 (23223073): 1754-176710.1038/nm.3012Crossref PubMed Scopus (634) Google Scholar, 2Evans J. Salamonsen L.A. Winship A. Menkhorst E. Nie G. Gargett C.E. Dimitriadis E. Fertile ground: human endometrial programming and lessons in health and disease.Nat. Rev. Endocrinol. 2016; 12 (27448058): 654-66710.1038/nrendo.2016.116Crossref PubMed Scopus (108) Google Scholar). These sequential events are controlled by the interplay of ovarian steroid hormones, estradiol (E2) and progesterone (P4) (3Fox C. Morin S. Jeong J.W. Scott Jr., R.T. Lessey B.A. Local and systemic factors and implantation: what is the evidence?.Fertil. Steril. 2016; 105 (26945096): 873-88410.1016/j.fertnstert.2016.02.018Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). During the proliferative phase, human endometrial epithelial cells and stromal cells (ESCs) increase in response to E2, leading to a thickening of the stratum functionalis of the endometrium. After ovulation, under P4 dominance, the endometrium, which is regulated by autocrine, paracrine, and transcription factors, transitions from the proliferative to the secretory phase (4Patel B. Elguero S. Thakore S. Dahoud W. Bedaiwy M. Mesiano S. Role of nuclear progesterone receptor isoforms in uterine pathophysiology.Hum. Reprod. Update. 2015; 21 (25406186): 155-17310.1093/humupd/dmu056Crossref PubMed Scopus (150) Google Scholar). The endometrial transition leads to spontaneous decidualization of human ESCs. Decidualization co-occurs with functional and morphological changes in human ESCs (5Gellersen B. Brosens J.J. Cyclic decidualization of the human endometrium in reproductive health and failure.Endocr. Rev. 2014; 35 (25141152): 851-90510.1210/er.2014-1045Crossref PubMed Scopus (385) Google Scholar). Uterine natural killer (uNK) cells are the predominant leukocyte in normal human endometrium (6Nagler A. Lanier L.L. Cwirla S. Phillips J.H. Comparative studies of human FcRIII-positive and negative natural killer cells.J. Immunol. 1989; 143 (2530273): 3183-3191PubMed Google Scholar). Approximately 70–80% of uNK cells are characterized as CD56brightCD16− (7King A. Loke Y.W. On the nature and function of human uterine granular lymphocytes.Immunol. Today. 1991; 12: 432-43510.1016/0167-5699(91)90014-KAbstract Full Text PDF PubMed Scopus (265) Google Scholar). Activated uNK cells can produce angiogenic factors, which promote spiral artery remodeling, and secrete cytokines that direct the migration and invasion of the trophoblast by interaction with trophoblast surface antigens (8Jokhi P.P. King A. Sharkey A.M. Smith S.K. Loke Y.W. Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction.J. Immunol. 1994; 153 (7525703): 4427-4435PubMed Google Scholar, 9Dosiou C. Giudice L.C. Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives.Endocr. Rev. 2005; 26 (15689572): 44-6210.1210/er.2003-0021Crossref PubMed Scopus (289) Google Scholar, 10Kalkunte S.S. Mselle T.F. Norris W.E. Wira C.R. Sentman C.L. Sharma S. Vascular endothelial growth factor C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetal interface.J. Immunol. 2009; 182 (19299706): 4085-409210.4049/jimmunol.0803769Crossref PubMed Scopus (148) Google Scholar). Importantly, the uNK cells and other leukocytes in the endometrium do not express P4 receptors (11Henderson T.A. Saunders P.T. Moffett-King A. Groome N.P. Critchley H.O. Steroid receptor expression in uterine natural killer cells.J. Clin. Endocrinol. Metab. 2003; 88 (12519888): 440-44910.1210/jc.2002-021174Crossref PubMed Scopus (217) Google Scholar). The activation and survival of uNK cells have been associated with the decidual-specific factor, interleukin-15 (IL15), in the human endometrium (12Kitaya K. Yamaguchi T. Honjo H. Central role of interleukin-15 in postovulatory recruitment of peripheral blood CD16(−) natural killer cells into human endometrium.J. Clin. Endocrinol. Metab. 2005; 90 (15713701): 2932-294010.1210/jc.2004-2447Crossref PubMed Scopus (83) Google Scholar). Previously, we showed that one transcription factor, heart and neural crest derivatives-expressed transcript 2 (HAND2), was up-regulated by E2 and a representative progestin, medroxyprogesterone (MPA), in a time- and dose-dependent manner for ESC decidualization (13Cho H. Okada H. Tsuzuki T. Nishigaki A. Yasuda K. Kanzaki H. Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells.Fertil. Steril. 2013; 99 (23036802): 248-25510.1016/j.fertnstert.2012.08.056Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Originally, HAND2 was identified as a basic helix-loop-helix transcription factor for embryonic right ventricles (14Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.Nat. Genet. 1997; 16 (9171826): 154-16010.1038/ng0697-154Crossref PubMed Scopus (541) Google Scholar, 15Yamagishi H. Olson E.N. Srivastava D. The basic helix-loop-helix transcription factor, dHAND, is required for vascular development.J. Clin. Invest. 2000; 105 (10675351): 261-27010.1172/JCI8856Crossref PubMed Scopus (116) Google Scholar). In human ESCs, silencing of HAND2 reduced both the morphological differentiation and the decidual-specific factors, including prolactin, forkhead box O1A (FOXO1A), interleukin-15 (IL15), fibulin-1, and tissue inhibitor of metalloproteinase-3 (16Shindoh H. Okada H. Tsuzuki T. Nishigaki A. Kanzaki H. Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.Fertil. Steril. 2014; 101 (24745730): 1781-1790.e1-510.1016/j.fertnstert.2014.03.013Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Therefore, it is conceivable that IL15 transcription is directly or indirectly regulated by HAND2. To the best of our knowledge, no studies have evaluated transcription factors directly affecting IL15 in human ESCs. We have applied in silico analysis here to identify HAND2 motifs (CHTCTGG) (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) and found candidate sequences in the upstream and intronic region of the human IL15 locus. The present study aimed to examine whether HAND2 participates in IL15 transcriptional regulation in human ESCs. First, we examined whether HAND2 and IL15 were up-regulated in the human endometrium as well as cultured ESCs. During the secretory phase (days 15–28 from previous menstruation, n = 12), HAND2 and IL15 were significantly increased compared with the proliferative phase (days 6–11 from previous menstruation, n = 8) (HAND2: 6.08 ± 5.40 (mean ± S.D.) versus 2.00 ± 0.73, p < 0.05; IL15: 6.86 ± 4.63 versus 2.47 ± 1.34, p < 0.05) (Fig. 1). There is a significant correlation between HAND2 and IL15 in the human endometrium (r2 = 0.4239, p < 0.05) (Fig. 1). In the endometrium sections (Fig. 2), pseudostratification of the nuclei in the glandular epithelium was observed as the proliferative phase unfolded. In the secretory phase, glandular epithelium transformed to a secretory form in the glands as monolayer cubic epithelium. ESCs in the endometrium transformed from fibroblast-like in the proliferative phase to epithelium-like with cytoplasmic expansion, large pale nuclei, and rounding of cells in the secretory phase as predecidua. Similar to quantitative PCR (qPCR) results, histological analysis indicated that HAND2 protein and IL15 mRNA increased in the secretory phase in the endometrium compared with the proliferative phase (Fig. 2). Although both are estimated to be expressed in ESCs, HAND2 and IL15 mRNA were observed in many, but not all, ESCs in the endometrium during the secretory phase (Fig. 2). HALO analysis shows that during the secretory phase, there are 390.7 ± 50.3 (312–464) HAND2-positive cells/0.1 mm2 (Fig. S1). Cells with IL15 signals exhibited 100% merge with HAND2 signals in the secretory phase. Conversely, cells with HAND2 signals did not always merge with IL15 signals; the results demonstrated that 60.3 ± 6.4 (48.9–70.3)% of HAND2-positive cells were merged with the IL15, suggesting a time lag for IL15 transcription start by HAND2. Similar regions within 2 kbp upstream regions from transcription start sites between human IL15 and mouse, rat, or monkey IL15 gene were analyzed using VISTA (18Mayor C. Brudno M. Schwartz J.R. Poliakov A. Rubin E.M. Frazer K.A. Pachter L.S. Dubchak I. VISTA: visualizing global DNA sequence alignments of arbitrary length.Bioinformatics. 2000; 16 (11159318): 1046-104710.1093/bioinformatics/16.11.1046Crossref PubMed Scopus (736) Google Scholar, 19Frazer K.A. Pachter L. Poliakov A. Rubin E.M. Dubchak I. VISTA: computational tools for comparative genomics.Nucleic Acids Res. 2004; 32 (15215394): W273-W27910.1093/nar/gkh458Crossref PubMed Scopus (1313) Google Scholar) (Fig. S2). IL15 upstream regions between humans and mice or humans and rats are not conserved, but regions between humans and monkeys are well-conserved. Among them, the positions from −439 to −1 in the human IL15 upstream region are not conserved (Fig. S2). Next, a homology search among Homo sapiens, Macaca fascicularis, and Mus musculus was performed using ClustalW version 2.1 (Fig. 3A). We found the putative consensus sequence for HAND2 binding (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) in the well-conserved upstream region and intronic regions of human IL15 locus using in silico analysis. Putative HAND2 motifs are denoted as CCTCTGG at position −1628/−1622, relative to the transcription start site, in the upstream region of the human IL15 gene (Fig. 3, A and B). One putative HAND2 motif was also found in the first intron of the human IL15 locus and is denoted as CATCTGG at position intervening sequences (IVS)1_16239/16245 (Fig. 3B). Introns have been shown to affect gene expression by a number of known and unknown mechanisms, collectively known as intron-mediated enhancement (IME) (20Mascarenhas D. Mettler I.J. Pierce D.A. Lowe H.W. Intron-mediated enhancement of heterologous gene expression in maize.Plant Mol. Biol. 1990; 15 (2103480): 913-92010.1007/BF00039430Crossref PubMed Scopus (184) Google Scholar). However, introns must be within 1 kbp of the transcription start to exert an effect via IME. Because the length of exon 1 in the human IL15 gene is 153 nucleotides, it has been suggested that the putative HAND2 motif at IVS1_16239/16245 is involved in IME. Therefore, ChIP-PCR analysis was performed using primers specific to the human IL15 upstream region around the upstream putative motif, the putative motif in intron 1, negative control primers (pair −1110F and −933R and pair −1937F and −1761R) (Fig. 3B), and immunoprecipitated purified DNA. ChIP-PCR analysis using the −1687F and −1515R primer pair revealed a positive reaction for the human IL15 upstream region around the putative HAND2 motif (Fig. 3C). An additional positive reaction was found in the experiment using the −1649F and −1405R primer pair (Fig. 3C). PCR against the putative HAND2 motif included in intron 1 with the IVS1_16153F and IVS1_16346R primer pair or the IVS1_16002F and IVS1_16253R primer pair showed no positive amplifications. No amplification was detected from purified DNA incubated with normal rabbit IgG (Fig. 3C) or water, which were used as negative control templates, or negative control experiments with the −1110F and −933R primer pair and the −1937F and −1761R primer pair (Fig. 3C). We also confirmed the binding of HAND2 to the HAND2 motifs at −1628/−1622 using ChIP-qPCR with ESC immunoprecipitated chromatin samples from three independent patients (Fig. 3D). Even in ChIP-qPCR, HAND2 recognized the CCTCTGG sequence at position −1628/−1622 of the IL15 upstream region without the CATCTGG at position IVS1_16239/16245 and the negative control region (Fig. 3D). Subsequently, to clarify the significance of the 1.8-kbp upstream region, including the position at −1628/−1622 of the IL15 gene on IL15 transcription in ESCs, we performed a luciferase reporter assay with IL15ups/pGL4.10 (Fig. 4A) under treatment with E2 and MPA. No significant responses were observed between pGL4.10 with/without E2 and MPA treatment (1.19 ± 0.09/1.00 ± 0.38) (Fig. 4B), indicating an absence of confounding ESCs endogenous transcriptional regulatory elements in the pGL4.10 plasmid. Additionally, there are no significant responses between pGL4.10 and IL15ups/pGL4.10 without treatment (1.35 ± 0.35), but E2 and MPA treatment significantly increased luciferase activity of IL15ups/pGL4.10 (6.78 ± 1.06, p < 0.05) (Fig. 4B) compared with no treatment. Even in ESCs, from a different patient with the estimated secretory phase, E2 and MPA stimulation significantly increased luciferase activity (18.79 ± 2.20, p < 0.05; Fig. S3) compared with no stimulation. It is suggested that the upstream region of the IL15 gene can regulate IL15 transcription in ESCs under E2 and MPA stimulation in isolation, without intronic sequence. Alternatively, in this different patient's ESCs, IL15ups/pGL4.10 showed significant elevation in luciferase activities without E2 and MPA stimulation (5.47 ± 0.67, p < 0.05; Fig. S3) compared with pGL4.10 without E2 and MPA stimulation (1.00 ± 0.06) (Fig. S3), suggesting the presence of an endogenous transcription factor for IL15 prior to E2 and MPA stimulation in this ESCs. Thereafter, we examined whether HAND2 regulates IL15 transcription via the 1.8-kbp upstream region of IL15, including the position at −1628/−1622, in ESCs. Co-transfection of the pGL4.10 plasmid with the pIRES2-AcGFP1 (1.00 ± 0.36) or HAND2/pIRES2 (1.26 ± 0.42) vectors exhibited similar luciferase expression in ESCs (Fig. 4C). Luciferase activity from IL15ups/pGL4.10 with the pIRES2-AcGFP1 vector (mock) exhibited no significant increase compared with the pGL4.10 and pIRES2-AcGFP1 vector (mock) (1.27 ± 0.26) in ESCs (Fig. 4C). Activity in the IL15ups/pGL4.10 group increased significantly via co-transfection with the HAND2/pIRES2 vector compared with mock in ESCs (2.76 ± 0.88; p < 0.05; Fig. 4C). Subsequently, the luciferase activity of deletion (ΔH2_motif/pGL4.10) (Fig. 4A) and substitution (CTGtoGAC/pGL4.10) (Fig. 4A) mutants of the putative HAND2 motif were examined. The luciferase activity from IL15ups/pGL4.10 with HAND2/pIRES2 (3.25 ± 0.85) was significantly up-regulated in estimated secretory phase ESCs from different patients compared with pIRES2-AcGFP1 (1.64 ± 0.51, p < 0.05) (Fig. 4D). These results indicate that there is no individual difference in HAND2-induced IL15 up-regulation in each ESC series. Conversely, the ΔH2_motif/pGL4.10 (2.05 ± 0.65, p < 0.05) or CTGtoGAC/pGL4.10 (2.08 ± 0.74, p < 0.05) with pIRES2-AcGFP1 showed significant promoter activity compared with pGL4.10 with pIRES2-AcGFP1 (1.00 ± 0.25) (Fig. 4D). There are no responses against HAND2/pIRES2 in the ΔH2_motif/pGL4.10 (1.80 ± 0.30) and CTGtoGAC/pGL4.10 (1.74 ± 0.39). To examine whether HAND2 effect against IL15 transcription is specific in ESCs, we conducted a reporter assay using IL15ups/pGL4.10 in HEK293T cells (Fig. S4). Although the IL15ups/pGL4.10 showed higher luciferase activities in HEK293T cells than in ESCs, no reactivity was observed in the 1.8-kbp IL15 upstream region against HAND2 in HEK293T cells (Fig. S4). This suggests that HAND2 effect against IL15 transcription might be specific in ESCs, or the 1.8-kbp IL15 upstream region might be suppressed, and this suppression might be released by HAND2 in ESCs. In our previous siRNA experiments against HAND2 (16Shindoh H. Okada H. Tsuzuki T. Nishigaki A. Kanzaki H. Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.Fertil. Steril. 2014; 101 (24745730): 1781-1790.e1-510.1016/j.fertnstert.2014.03.013Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), FOXO1 expression was synchronized with HAND2 expression. This may suggest that FOXO1 transcription is regulated by HAND2 and that FOXO1 coordinately regulates IL15 transcription in ESCs. We have also identified putative FOXO1 motifs (TGTTTT) (21Rose A.B. Elfersi T. Parra G. Korf I. Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression.Plant Cell. 2008; 20 (18319396): 543-55110.1105/tpc.107.057190Crossref PubMed Scopus (121) Google Scholar, 22Osterwalder M. Speziale D. Shoukry M. Mohan R. Ivanek R. Kohler M. Beisel C. Wen X. Scales S.J. Christoffels V.M. Visel A. Lopez-Rios J. Zeller R. HAND2 targets define a network of transcriptional regulators that compartmentalize the early limb bud mesenchyme.Dev. Cell. 2014; 31 (25453830): 345-35710.1016/j.devcel.2014.09.018Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Mackay L.K. Wynne-Jones E. Freestone D. Pellicci D.G. Mielke L.A. Newman D.M. Braun A. Masson F. Kallies A. Belz G.T. Carbone F.R. T-box transcription factors combine with the cytokines TGF-β and IL-15 to control tissue-resident memory T cell fate.Immunity. 2015; 43 (26682984): 1101-111110.1016/j.immuni.2015.11.008Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 24Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339 (23287718): 819-82310.1126/science.1231143Crossref PubMed Scopus (8346) Google Scholar) in the upstream region of human IL15 gene (positions at −1131/−1137 and −1993/−1998). Therefore, we examined whether IL15 was indeed regulated by FOXO1 in ESCs. ChIP-qPCR analysis was performed using primers to the putative FOXO1 motif at −1131/−1137 (Fig. 3B) and at −1993/−1998 (Fig. S5) and immunoprecipitated purified DNA. ChIP-qPCR analysis using the −1286F and −1114R, −2065F to −1916R, and −2051F to −1907R primer pairs showed no significant difference between the immunoprecipitated-DNA with normal rabbit IgG and FOXO1 antibody (Fig. 3D and Fig. S5), indicating no recruitment of FOXO1 to the IL15 upstream region in our ESCs as reported in previous ChIP-sequencing analysis with FOXO1 antibody and decidualizing ESCs (25Vasquez Y.M. Mazur E.C. Li X. Kommagani R. Jiang L. Chen R. Lanz R.B. Kovanci E. Gibbons W.E. DeMayo F.J. FOXO1 is required for binding of PR on IRF4, novel transcriptional regulator of endometrial stromal decidualization.Mol. Endocrinol. 2015; 29 (25584414): 421-43310.1210/me.2014-1292Crossref PubMed Scopus (49) Google Scholar). Because it has been reported that FOXO1 binds to human IGFBP1 enhancer region in ESCs with decidualization (26Tamura I. Jozaki K. Sato S. Shirafuta Y. Shinagawa M. Maekawa R. Taketani T. Asada H. Tamura H. Sugino N. The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.J. Biol. Chem. 2018; 293 (29453285): 5270-528010.1074/jbc.RA117.000234Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), as a control experiment, we conducted ChIP-qPCR with a primer pair for the IGFBP1 enhancer region (Table S1). FOXO1 antibody significantly immunoprecipitated the human IGFBP1 enhancer region, including in our ESCs (Fig. S5), indicating the effect of FOXO1 antibody. FOXO1 protein was indeed expressed in this study and was recruited to IGFBP1 upstream region under our E2 and MPA treatment. To further confirm the FOXO1 effects against 1.8 kbp of the IL15 upstream region, we performed a reporter assay. HAND2 significantly increased the luciferase activities even in the 1.8-kbp IL15 upstream region in these new ESCs obtained from a distinct patient; however, there is no reactivity found against FOXO1 (Fig. S6). We also examined the possibility of FOXO1 functioning as a repressor in the IL15 upstream region but found no effect of FOXO1 as the variants in the FOXO1 motif had no reactivity against FOXO1 (Fig. S6). Unlike HAND2, FOXO1 is unlikely to be involved in the transcriptional modulation of the 1.8-kbp upstream region of IL15 following E2 and MPA stimulation. This report is the first to clearly demonstrate that HAND2 is a transcription factor that directly regulates IL15 expression in human ESCs. We have identified a CCTCTGG sequence in the proximal region of the human IL15 gene as a putative HAND2 motif using ChIP-PCR analysis and a luciferase reporter assay, using a human IL15 upstream region and its mutants in ESCs. Our results suggest a functional connection between HAND2 and this putative HAND2 motif (CCTCTGG) in regulating human IL15 transcription. We did not find any functional association between FOXO1 and IL15 expression as reported previously (25Vasquez Y.M. Mazur E.C. Li X. Kommagani R. Jiang L. Chen R. Lanz R.B. Kovanci E. Gibbons W.E. DeMayo F.J. FOXO1 is required for binding of PR on IRF4, novel transcriptional regulator of endometrial stromal decidualization.Mol. Endocrinol. 2015; 29 (25584414): 421-43310.1210/me.2014-1292Crossref PubMed Scopus (49) Google Scholar). VISTA analysis showed no significant similar region between rodents and primates with a criterion of 70% per 100 nucleotides. Unlike the human endometrium, rodent endometrium does not induce thickening of the stratum functionalis without embryo implantation. In actuality, decidualization of the endometrium is under maternal control in a handful of species, which includes higher primates (humans, apes, and Old World monkeys), some bats, and the elephant shrew (5Gellersen B. Brosens J.J. Cyclic decidualization of the human endometrium in reproductive health and failure.Endocr. Rev. 2014; 35 (25141152): 851-90510.1210/er.2014-1045Crossref PubMed Scopus (385) Google Scholar). Therefore, the upstream regions are not conserved between rodents and primates, and whole transcriptional regulation of IL15 might be distinct among them. However, because ClustalW multiple alignment indicated that the HAND2 motif in the upstream region of the IL15 gene is well-conserved (Fig. 3C), the relationship between HAND2 and IL15 transcription in ESCs might be conserved even in rodents. A chimeric motif is supposed to have been generated by connecting forward and backward sequences around the putative HAND2 motif in the deletion mutant. Further, the deletion mutant altered positional relations of all known and unknown motifs in the upstream region of the IL15 gene. For instance, we previously found that nuclear receptor subfamily 6, group A, member 1 (NR6A1), a DNA-binding factor, down-regulates hypocretin expression through an 18-bp nuclear receptor response element (NurRE) in the upstream region of the hypocretin gene (27Tanaka S. Kodama T. Nonaka T. Toyoda H. Arai M. Fukazawa M. Honda Y. Honda M. Mignot E. Transcriptional regulation of the hypocretin/orexin gene by NR6A1.).Biochem. Biophys. Res. Commun. 2010; 403 (21056546): 178-18310.1016/j.bbrc.2010.11.001Crossref PubMed Scopus (12) Google Scholar). We then cloned a single copy or three copies of NurRE into the upstream region of the TATA box in pTAL-luc reporter plasmid to investigate whether the NurRE exerted transcriptional modulator activity (enhancer, silencer, or insulator). In the condition with the NR6A1 expression vector, three copies were surprisingly activated, although a single copy was repressed. This switching mode might be induced by a generated chimeric binding site connecting each NurRE to other motifs or the change with proximity of NurRE to the promoter. Therefore, we have examined the activities of not only a deletion mutant, but also a substitution mutant, to confirm that the putative HAND2 motif in the human IL15 transcription functions appropriately in ESCs. CTG changed to GAC at −1623/−1625 (purine base to pyrimidine base or pyrimidine base to purine base), but the length from the transcription start site and locations of the other putative motifs might not differ in this substitution mutant. Among these examinations, both mutants showed no response against HAND2. Therefore, HAND2 could regulate IL15 transcription via the putative HAND2 motif at −1628/−1622 in the upstream region of the IL15 gene in ESCs. A recent single-cell analysis reported that a subclass of ESCs express IL15, ruling out IL15 expression by all ESCs (28Vento-Tormo R. Efremova M. Botting R.A. Turco M.Y. Vento-Tormo M. Meyer K.B. Park J.-E. Stephenson E. Polański K. Goncalves A. Gardner L. Holmqvist S. Henriksson J. Zou A. Sharkey A.M. et al.Single-cell reconstruction of the early maternal-fetal interface in humans.Nature. 2018; 563 (30429548): 347-35310.1038/s41586-018-0698-6Crossref PubMed Scopus (491) Google Scholar). Therefore, two possibilities exist—either HAND2 transcription is different in each ESC, or HAND2 is expressed at a similar level in all ESCs, but the susceptibility (epigenetic alterations, such as methylation) of the IL15 locus against HAND2 is different for each ESC. Our immunohistochemical results show that HAND2 is not expressed in all ESCs; therefore, HAND2 may be regulated by different mechanisms in each ESC. Interestingly, lack of expression of IL15 might indicate no requirement of uNK around them, suggesting that a local prevalence of uNK cells could trigger IL15 expression. HAVCR2 (also known as TIM3)-LGALS9, a factor associated w
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