Folate receptor alpha upregulates Oct4, Sox2 ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells VINEET MOHANTY,a AMAR SHAH,a ELISE ALLENDER,a M. RIZWAN SIDDIQUI,a SARAH MONICK,a SHUNSUKE ICHI,b BARBARA MANIA-FARNELL,c DAVID G. MCLONE,a TADANORI TOMITA,a CHANDRA SHEKHAR MAYANILa Key Words. Cranial neural crest cells • Folate receptor alpha • Oct4 • Sox2 • Klf4 • Enhancer/ promoters • Multipotency • miR-let-7 • miR-138

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Developmental Biology Program, Stanley Manne Children’s Research Institute, Division of Pediatric Neurosurgery, Ann and Robert H. Lurie Children’s Hospital of Chicago, Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA; b Department of Neurosurgery, Japanese Red Cross Medical Center, Shibuya-Ku, Tokyo, Japan; cDepartment of Biology, Purdue University at Calumet, Hammond, Indiana, USA

Correspondence: Chandra Shekhar Mayanil, Ph.D., Developmental Biology Program, 2430 N Halsted Street, M/c 204, Stanley Manne Children’s Research Institute, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, Illinois 60614, USA. Telephone: 1-773-755-6530; Fax: 1-773-755-6385; e-mail: [email protected] Received December 30, 2015; accepted for publication May 28, 2016; first published online in STEM CELLS EXPRESS June 14, 2016. C AlphaMed Press V

ABSTRACT Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRa) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRa translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRa regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRa upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRa upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-50 enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRa downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRa interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into nonproliferating neural crest cells (NCCs) derived from Splotch (Sp2/2), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRa: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. STEM CELLS 2016; 00:000–000

SIGNIFICANCE STATEMENT The glycosylphosphatidylinositol (GPI) anchored folate receptor alpha (FRa) plays an important role in neural crest and neural tube formation during embryonic development. In previous studies our lab reported that FRa translocates to the nucleus upon folate binding, where it acts as a transcription factor. Here we report the pleiotropic role of FRa in that it: (a) upregulates genes responsible for pluripotency, such as Oct4, Sox2, and Klf4, by binding to cis-regulatory enhancer/promoter regions, and (b) downregulates miR-138 (which target Oct4) and miRlet-7 (which target Trim71). These novel functions of FRa at an early developmental time point provide mechanisms that explain why it is critical to supplement with FA prior to pregnancy, to obviate neural tube defects (NTDs). In addition, FRa regulation of miR-138/Oct4/Trim71 and miR-let-7/Trim71 axis may help explain how pre-migratory neural crest cells maintain their multipotent phenotype and their proliferation potential prior to differentiation.

1066-5099/2016/$30.00/0 http://dx.doi.org/ 10.1002/stem.2421 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

INTRODUCTION Folic acid (FA) prevents NTDs and thus is a highly recommended nutritional supplement for women of child bearing age. One of the questions in developmental biology is to understand the mechanism behind this action of FA. Previous work from our laboratory demonstrated that neural crest stem cells (NCSCs) cultured from neural tubes (NTs) of Splotch homozygous (Sp2/2 or Pax32/2) embryos

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exhibit open NTs, did not proliferate or migrate and differentiate prematurely [1]. Maternal supplementation to Sp1/2 female mice with FA restored proliferation potential of neural stem cells [2, 3]. This raised the question: how does FA maintain proliferation potential and the multipotent character of NCSCs prior to differentiation? Answering this question is crucial in that NCSC generate diverse lineages including peripheral neurons, glia, melanocytes, and mesenchymal

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Novel Functions of Folate Receptor Alpha

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derivatives. Understanding the mechanisms of NCC proliferation, migration and differentiation will help determine how these cells work and allow scientists to use them as models for NC-associated disorders and ultimately find innovative molecular target for clinical treatments. Two mechanisms exist for cellular uptake of FA: (a) low affinity mechanism comprising of proton-coupled folic acid transporter, and reduced folic acid carrier and (b) a high affinity mechanism involving a folate receptor alpha (FRa, also known as Folbp1 or Folr1), which is linked to cell surfaces via a glycosylphosphatidylinositol anchor [4]. FRa is critical for embryonic development, where it is involved in NCC and NT formation in addition to other functions [5–9]. Disruption of FRa expression and function causes early NCC migration associated craniofacial anomalies, abnormal heart development, and NTDs [10]. Nullizygous FRa embryos (Folbp12/2) die in utero by gestational day E10. Affected genes in these embryos include transcription factors, G-proteins, growth factors, methyltransferases, and genes involved in cell proliferation [5–8]. FRa translocates to the nucleus in response to FA binding, where it acts as a transcription factor for Pax3 downstream targets, such as Hes1 and Fgfr4 [11]. We hypothesize that FRa may also act as a transcription factor at pluripotency genes found in NCCs. NCCs are “multipotent” not “pluripotent” [12], however they express pluripotency genes, although at low levels compared to embryonic stem cells. These include Oct4, Lin28, Sox2, Nanog, SSEA-1, SSEA-3, SSEA4, TRA-1-60, and TRA-1-81 [12, 13]. This begs the question, does FRa regulate genes responsible for pluripotency, such as Oct4, Sox2, Klf4, and Nanog to maintain “stem-like” characteristics of NCCs while they are migrating. Another group of molecules which regulates NC development are miRNAs [14–16]. In mammals, miRNAs, 22 nucleotide long noncoding RNAs, play critical roles in various physiological processes by acting as post-transcriptional regulators to reduce translation of target genes by either destabilizing mRNAs or blocking their translation [17, 18]. In the NC, repression of targets by regional and spatio-temporal biogenesis of miRNAs elicits critical changes in gene expression programs that regulate gene regulatory networks crucial to NC development [19]. Zehir at al. [20] have shown that loss of Dicer in the head region leads to a loss of NC-derived craniofacial bones. Loss of Dicer did not prevent initial differentiation of NC, but as development progressed NC derivatives were lost due to apoptotic cell death suggesting that Dicer and miRNAs are required for survival of NC-derived tissues by preventing apoptosis during differentiation [20]. In a previous study, we found twelve miRNAs upregulated in Sp-/- (Pax3-/-) embryos displaying NTDs. Pre-conceptual FA supplementation to Sp1/- female mice normalized the levels of these miRNAs in FA rescued Sp-/- (Pax3-/-) embryos [2]. This suggests that: (a) Pax3 may negatively regulate these miRNA levels in wild type embryos, which are then elevated in the absence of functional Pax3; and (b) FA through FRa may negatively regulate these miRNAs in FA rescued Sp-/- embryos. To investigate this concept, this study examined levels of miR138 and miR-let-7 in response to treatment with FA in NCCs. These two miRNAs are among the 12 upregulated in Sp-/embryos [2] and they target Oct4 (miR-138) and one of its effectors Trim71 (miR-let-7) [21].

Thus, our investigation tested the hypotheses that FRa upregulates pluripotency markers in CNCCs by binding to their enhancer/promoter regions while simultaneously downregulating miRNAs associated with these markers, specifically the miRNAs that downregulate Oct4 and its downstream effector target Trim71 [21]. The results presented suggest a novel pleiotropic role of FRa: (a) direct activation of Oct4, Sox2, and Klf4 genes by binding to their cis-regulatory elements; and (b) repression of miRNA biogenesis, miR-138 and miR-let-7, possibly by interfering with the activity of the Drosha/DGCR8 complex.

MATERIALS

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METHODS

Reagents and Animals A list of primary and secondary antibodies used in this study and PCR primers and oligonucleotides used for electromobility shift assay (EMSA) are mentioned in Supporting Information Table S1 and S2 (Excel file). All animal experiments were approved by IACUC–Stanley Manne Children’s Research Institute (Approval #Mayanil, IACUC ID: 13-001.0 09) and performed in accordance with institutional guidelines and regulations.

Cranial Neural Crest Cell (CNCCs) Culture CNCC cell line O9-1 obtained from Wnt1-Cre; R26R-GFP [22–25] from E8.5 mouse embryos was kindly provided as a gift by Dr. Robert E Maxon Jr (Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA). Basal medium for NC culture was prepared as described by Ishii et al. [23].

Real Time Quantitative RT-PCR Real time quantitative RT-PCR was done as described previously [26]. Primers and probes used in this study were designed using Primer Express software (PerkinElmer Life Sciences, Naperville, IL, http://www.perkinelmer.com/corporate/ what-we-do/markets/life-sciences). Primers were synthesized by Eurofins (Louisville, KY, http://www.eurofinsgenomics.com/ en/products/dnarna-synthesis/oligo-options.aspx). Refer to Supporting Information Table S1 for primers.

Quantitative RT-PCR for miRNA Total RNA was isolated by using miRNeasy kit (Qiagen, Valencia, CA, https://www.qiagen.com/us/shop/sample-technologies/rna/rna-preparation/mirneasy-mini-kit#orderinginformation). Quantitative RT-PCR was done (as per manufacturer’s instructions, Applied Biosystems (Grand Island, NY, http:// www.thermofisher.com/us/en/home/life-science/pcr/real-timepcr/real-time-pcr-assays.html) to identify changes in miRNA expression. Briefly, 50 ng of total RNA was reversetranscribed using the TaqMan miRNA Reverse Transcription Kit with primers for let-7f and miR-138 and U6 small nuclear RNA (Applied Biosystems). This was followed by RT PCR with a standard TaqMan microRNA assay protocol, with TaqMan probes for these miRNAs. The relative expression of each miRNA was determined in reference to an internal U6 small nuclear RNA control.

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Chromatin Immunoprecipitation (ChIP) Assays ChIP assays were performed as described previously [11, 27]. PCR was performed with primers for murine Oct4, Sox2, Klf4, and Nanog enhancer/promoter regions (Supporting Information Table S1 for primers). All ChIP samples were tested for false-positive PCR amplification by sequencing the amplified product to ascertain the specificity of FRa binding to cis-regulatory elements.

Electro-Mobility Shift Assays Probes were prepared for EMSA by annealing complementary oligonucleotides representing selected regions of murine Oct4, Sox2, and Klf4 50 enhancer/promoter regions followed by 50 end labeling with [g-32P] ATP by T4 polynucleotide kinase. EMSA was done as described previously [11]. For shift assays 100 ng nuclear protein extract from FA treated CNCCs was pre-incubated at room temperature for 30 minutes in EMSA reaction buffer (100 mM Tris-HCl pH7.5, 500 mM KCl, 6.5% glycerol, 50 mM pyrophosphate, 25 mM DTT in 2.5% Tween 20, 1 mg/ml salmon sperm DNA) in the presence of 32P labeled oligonucleotides. For cold reactions the nuclear protein was pre-incubated with 100-fold molar excess of unlabeled oligonucleotides for 30 minutes prior to adding labeled oligonucleotides. For super-shift, FRa antibody (10 ng) was incubated with 100 ng nuclear extract for 4 hours at room temperature and then oligonucleotides were added with the EMSA reaction buffer for an additional 30 minutes at RT. After incubation, free DNA and DNA protein complexes were resolved in 6% polyacrylamide gels. Electrophoresis was performed at 1000 V and 25 mA for 2 hours in a continuous cooling system. To visualize shifted or super-shifted bands, gels were dried at RT and transferred to Phosphor Imager Screens (Amersham Biosciences, Pittsburgh, PA, http://www. gelifesciences.com). Gels were exposed overnight at RT.

FRa Knockdown FRa-shRNA constructs TG304479A, TG304479B, TG304479C, TG304479D, and scrambled shRNA construct TR30013 (OriGene, Rockville, MD, http://www.origene.com), were transfected in cranial NCCs using Lipofectamine3000 (Invitrogen, Grand Island, NY, https://www.thermofisher.com/us/en/home/ brands/invitrogen.html) in a six well format as per manufacturer’s instructions. Forty-eight hours post-transfection the expression of FRa level was ascertained by western blot. The construct, TG304479B was most effective in knocking down the FRa expression and hence used in all subsequent experiments. In experiments (as in Figs. 1, 4), the cells were treated with FA 48 hours post transfection. To maintain FRa knockdown, the cells (for 48 hours time point) were re-transfected after 24 hours of initial FA treatment.

Oct4 and Trim71 Knockdown piSicoR-mCh-Oct4i construct (a gift from Dr. Miguel RamalhoSantos (Addgene plasmid # 21906, Cambridge, MA, http:// www.addgene.org)) and Trim71 or LIN-41 sh-RNA plasmid (sc72329-SH) (Santa Cruz Biotechnology, Dallas, Texas, www.scbt. com) were transfected in cranial NCCs using Lipofectamine3000 (Invitrogen) in a 6 well format as per manufacturer’s instructions.

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30 UTR-Trim71- and 30 UTR-Oct4-Renilla Luciferase Reporter Assays Putative miRNA binding sequences from miR-let-7 and miR-138, to the 30 UTR of Trim71 and Oct4, respectively, for the Renilla Luciferase reporter containing vector were custom synthesized by GeneCopoeia Inc (Rockville, MD, www.genecopoeia.com). The reporter vector without a miRNA binding sequences was used as a control. 30 -UTR-luciferase reporter constructs were transfected into cranial NCCs using the MegaTran 1.0 Transfection Reagent (Ori-Gene), with or without co-transfection of a shFolr1 vector (Ori-Gene). Firefly luciferase plasmid in pGL3 vector (5 ng/well) was used as an insertional control for transfection efficiency. Luciferase assays were performed using the Dual Luciferase kit (Promega, Madison, WI, www.promega.com).

Anti-miRNA Transfection Transfection with hsa-let-7f-2-3p mirVana miRNA inhibitor, Cat no. 4464084, ID: MH12370 (Ambion) hsa-let-7f-5p mirVana miRNA inhibitor, Cat no. 4464084, ID: MH10902, (Ambion, Grand Island, NY, https://www.thermofisher.com/us/en/home/ brands/invitrogen.html), hsa-miR-138-5p mirVana miRNA inhibitor, Cat no. 4464084, ID: MH11727, (Ambion) and scrambled Anti-miR miRNA Inhibitor Negative Control #1 Catalog number: AM17010 (Ambion) was performed using Lipofectamine RNAiMAX transfection reagent (Life Technologies, cat. no. 13778075). Cells were seeded to 80% confluency prior to transfection. For transfection, anti-miRNA-lipid complexes were formed as per the protocol using Opti-MEM medium, Lipofectamine RNAiMAX reagent, and respective miRNA inhibitors. Per well, the final concentration of miRNA inhibitors added amounted to 5 pmol for cells on a 24-well plate, and 25 pmol for those on 6 well plates. After 48 hours of transfection, the cells were either lysed in RIPA buffer for immunoblots or total RNA isolated for miRNAs, miR-138, miR-let-7 or Oct4 or Trim71 quantification. TaqMan microRNA assay kit (Cat no: 4427975, ID: 002284, Assay Name: hsa-miR-138); (Cat no: 4427975, Assay ID: 000382, Assay Name: hsa-let-7f) and (Cat no: 4427975 Assay ID: 001973, Assay Name: U6 snRNA) from Applied Biosystems were used for miRNA quantified by q-RT-PCR.

miRNA Isolation and qPCR Isolation of miRNA from cells and tissue samples was performed using the miRNeasy Mini Kit (Qiagen, cat no. 217004). Subsequent cDNA generation was accomplished using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4366596). Following assembly of all components for cDNA synthesis, the reverse transcription reaction was carried out in a thermocycler with the following steps: 30 minutes incubation at 168C, 30 minutes incubation at 428C, 5 minutes incubation at 858C, and a final hold at 48C. TaqMan Universal Master Mix II, no UNG (Applied Biosystems, cat no. 4440040) was used in preparing reactions for qPCR. Thermal cycling conditioned involved an initial 10 minutes hold at 958C for enzyme activation, then 40 cycles each consisting of a denaturation step at 958C for 15 seconds and then an annealing/ extension step at 608C for 60 seconds. Data collection for each cycle was done at the completion of the annealing/ extension step. RT primers (for cDNA synthesis) and TM primers (for qPCR) for U6 snRNA control, hsa-let-7f, and hsa-



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Figure 1. FA/FRa upregulates Oct4, Sox2, Klf4 and Trim71 but not Nanog mRNA in cranial neural crest cells. (A): Representative western blots of total protein lysates of O9-1 cells treated with FA (100 ng/ml) for indicated time in hours immunoblotted with Trim71, Oct4, Sox2, Klf4, and b-actin antibodies (see Supporting Information Table S1, Excel file). The (*) indicated above the band suggest upregulation of the protein expression at that time point of FA treatment. (B): Different FRa-shRNA constructs were used for knocking down FRa expression as indicated and scrambled shRNA was used a negative control. TG304479B construct shows complete knockdown of FRa and was used in subsequent FRa knockdown experiments. (C): The cranial NCCs were transfected with FRa-shRNA and (D): scrambled-shRNA constructs (OriGene), and treated with FA (100 ng/ml) for 3–48 hours, 24 hours post-transfection. Following treatment RNA was extracted and reverse transcribed to cDNA. Quantitative RT-PCR was done using mouse Oct4, Sox2, Klf4, Nanog and Trim71 forward and reverse primers (Supporting Information Table S1). n 5 6 and each data point in triplicate. **, p < .005; ***, p < .0001. Abbreviations: FA, folic acid; FRa, folate receptor alpha; NCC, neural crest cells.

miR-138 were all obtained from Applied Biosystem’s TaqMan MicroRNA Assays database.

Neural Tubes Explant Cultures and Isolation of Cranial NCCs from E10.5 Sp-/- Embryos Neural fold explant cultures from the open neural tube portion of the cranial region of E10.5 Sp-/- embryos were grown in NeuroBasal medium supplemented with bFGF (20 ng/ml) and EGF (20 ng/ml) on Matrigel coated 24 well plates. Cranial NCCs were allowed to migrate for 140 hours on Matrigel. NT explants were then removed and NCCs were plated onto matrigel coated six well plates, transfected with scrambled control anti-miR, antimiR-let-7f2-3p, anti-miR-let-7f-5p and anti-miR138. 24 h post transfection the cells were lifted off from the matrigel plate with PAPAIN (Worthington; LK003176, Lakewood, NJ, http:// www.worthington-biochem.com), 4 U/ml at 378C for 5 minutes, and collected in NeuroBasal medium supplemented with bFGF (20 ng/ml) and EGF (20 ng/ml). Approximately 2 3 104 cells/ml were added to 2 ml of NeuroBasal medium plus growth factors and allowed to grow in suspension cultures on low binding six well plates for an additional 7 days to ascertain the growth of spheres or colonies. Another term currently used for premigratory NCSC colonies is “Crestospheres” [25].

Statistical Analysis Using GraphPad Prism 6.0c software, data were presented as the mean values 6 the SEM. Two group differences were determined by an independent Student’s t test and multiple comparisons by a one-way ANOVA test (including Bonferroni’s post-hoc multiple comparisons). For Figure 6C and 6D, n 5 9. A “p” value less than 05 was considered significant.

RESULTS This study tested two hypotheses: (a) FRa transcriptionally regulates Oct4, Sox2, Klf4, and Nanog (b) FRa negatively regulates miR138 and miR-let-7 levels, which target Oct4 and Trim71 (a downstream effector of Oct4).

FA/FRa Upregulates Oct4, Sox2, Klf4, and Trim71, a Downstream Effector of Oct4, but not Nanog in CNCCs To test the hypothesis that FRa transcriptionally regulates, Oct4, Sox2, Klf4, and Nanog, CNCCs (O9-1 cells) were grown in six well plates in the absence (folate depleted medium) or presence of FA (100 ng/ml) for 3–48 hours. At specified time points, 25 lg of total protein lysates in RIPA buffer were


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Figure 2. FRa directly binds to the cis-regulatory elements of Oct4, Sox2, and Klf4 in cranial neural crest cells. Chromatin immunoprecipitation assays were performed using FRa, H3K27Ac and p300 antibodies, immunoprecipitated DNA was amplified via RT-PCR using forward and reverse primers from 50 -untranslated regions encompassing enhancer or promoter regions (Supporting Information Table S1) of (A) Oct4; (B) Sox2; (C) Klf4. n 5 4 and data point in triplicate. *, p < .05; **, p < .005. Abbreviations: FA, folic acid; FRa, folate receptor alpha.

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Figure 3. Electro-mobility Shift Assays (EMSA). EMSA of binding reactions using nuclear extracts from FA treated cranial neural crest cells and 32P-labeled double-stranded oligonucleotides of Oct4, Sox2, and Klf4 from regions defined by chromatin immunoprecipitation assays that gave maximum fold enrichment of immunoprecipitated DNA compared to input (Supporting Information Table S1). Arrows show shifted and super-shifted bands. Abbreviations: FRa, folate receptor alpha.

immunoblotted using Oct4, Sox2, and Klf4 antibodies with bactin as a positive control. The results showed that Oct4, Sox2, and Klf4 protein expression increased around 24 hour of FA treatment. Sox2 expression decreased at 48 hour of FA treatment. Additionally we investigated whether FA effects on Oct4, Sox2, and Klf4 could change the expression of downstream effector genes. We chose to look at Trim71, a downstream effector of Oct4 which promotes embryonic stem cell proliferation [21]. Trim71 expression was significantly upregulated at 24 hours of FA treatment (Fig. 1A). To confirm whether the increase in the expression was at the level of FA/FRa mediated transcription, total RNA was extracted from a separate set of CNCCs grown in six well plates (folate depleted medium) or presence of FA (100 ng/ ml) for 3–48 hours in the presence of FRa-ShRNA or scrambled control shRNA (scr-Sh-RNA). Out of four FRa-shRNA constructs provided by the manufacturer, the TG304479B construct was most effective in knocking down the FRa expression as confirmed by western blots (Fig. 1B) and hence used in all subsequent FRa knock down experiments. RNA from specified time points were reverse transcribed to cDNA. Subsequently, Oct4, Sox2, Klf4, Nanog, and Trim71 (a downstream effector of Oct4) expression was determined by qPCR. Following FRa-shRNA transfection Oct4, Sox2, Klf4, and Trim71 did not show increased expression in response to FA treatment (Fig. 1C). However, following scr-shRNA transfection: (a) Oct4 increased significantly (p < .0001) at 12 hours; (b) Sox2 increased significantly (p < .005) at 24 and 48 hours; (c) Klf4 increased steadily from 12 (12 and 24 hours p < .005) to 48 hours (p < .0001) in response to FA; (d) Nanog was not affected by FA; and (e) Trim71 increased (p < .005) in response to FA (Fig. 1D). These results suggested that Oct4, Sox2, Klf4, Nanog, and Trim71 responded differentially to FA treatment and clearly demonstrated that the FA mediated increase in the expression of Oct4, Sox2, Klf4, and Trim71 was mainly through interaction with FRa.

FRa Directly Binds to cis-Regulatory Elements of Oct4, Sox2, and Klf4 The next question addressed was: does FRa upregulate Oct4, Sox2, and Klf4 by directly binding to cis regulatory elements? ChIP assays were performed using FRa antibody to pull down FRa bound chromatin from CNCCs treated with FA for 15 or 30 minutes, followed by PCR with forward and reverse primers spanning different areas of the 50 -untranslated region of the gene, including promoter and distal and proximal enhancer regions. Oct4: FRa binds region 1 (distal enhancer region) and region 4 (promoter region) of Oct4 (Fig. 2A) by 15 minutes, with significant binding levels seen 30 minutes after FA treatment. To confirm that these regions define enhancer and promoter areas respectively, ChIP was performed using H3K27Ac and p300 antibodies, as these molecules are associated with active transcription. H3K27Ac and p300 bound to regions 1 and 4 of the 50 -untranslated region of Oct4. Sox2: ChIP assays on the 50 -untranslated region of Sox2 (Fig. 2B) show that from a total of eight identified areas, FRa only bound region 6, at 15 and 30 minutes following treatment with FA. Region 6 also bound H3K27Ac and p300. Klf4: ChIP assays on the Klf4 50 untranslated region (Fig. 2C) showed significant FRa binding to region 4 (Klf4 promoter) after a 30 minutes treatment with FA. H3K27Ac, but not p300, bound to region 4. EMSA was used to further confirm direct binding of FRa to Oct4, Sox2, and Klf4 cis regulatory elements (Fig. 3) using oligonucleotides from within the regions defined by ChIP for individual genes (Supporting Information Table S1). This is consistent with previous data for Hes1 and Fgfr4 genes showing FRa interaction with AANTT regions [11]. In summary, the above data demonstrate upregulation of Oct4, Sox2, and Klf4 genes by direct binding of FRa to their cis-regulatory elements.


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Figure 4. FRa negatively regulates the miRNA levels that target Oct4 and Trim71 in cranial neural crest cells. (A): Quantitative RT-PCR showing miR-let-7 and miR-138 in neural tubes (NT) (from wild type) and open neural fold (from Sp-/-) E10.5 mouse embryonic cranial NT, grown in the absence or presence of 100 ng/ml FA for 24 hours. RNA was isolated from migrating NCCs and miR-138 and miR-let-7 levels were determined by q-PCR. (B): CNCCs (O19) were treated or not treated with FA (100 ng/ml) for 1, 6, 12, 24, and 48 hours, and total was RNA isolated. Expression levels of miR-138 and miR-let-7 which target Oct4 and Trim71 respectively were determined by qPCR. Left panel: cells without FRa-shRNA transfection. Right panel: cells transfected with FRa-shRNA construct. (C): 30 UTR-Oct4-renilla luciferase (left panel) and 30 UTR-TRIM71-renilla luciferase (right panel) activities in the absence or presence of FRa-shRNA construct in CNCCs treated or not treated with FA (100 ng/ml) for 24 hours. n 5 6 and each data point in triplicate. ***, p < .0001. Abbreviations: FA, folic acid; FRa, folate receptor alpha; NCCs, neural crest cells.

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In summary, the results in this section show that: (a) FRa mediate the action of FA by decreasing miR-138 and miR-let7; and (b) decreased miRNA levels in turn produced an increase in the levels of Oct4 and TRIM71, downstream targets of miR-138 and miR-let-7, respectively.

Figure 5. FRa associates with Drosha and DGCR8. Coimmunoprecipitation showing FRa associates with Drosha and DGCR8 (Pasha). Cells were treated with FA (200 ng/ml) for indicated times, nuclear extract was isolated and immunoprecipitated (IP) overnight with anti-mouse FRa antibody, followed by immunoblotting (IB) with Drosha and DGCR8 antibody, and FRa. Mouse IgG was used as a negative control. Abbreviations: FRa, folate receptor alpha.

FRa Negatively Regulates the miRNAs That Target Oct4 and Trim71 In previous studies maternal (Sp1/-) supplementation of FA rescued NTDs in Sp-/- embryos [2]. Rescue was accompanied by down-regulation of the levels of 12 miRNAs that were upregulated in Sp-/- NTD embryos. Of the 12 miRNAs, miR-let7 targets Trim71 and miR-138 targets Oct4; (www.microRNA. org; and http://www.targetscan.org). To test the hypothesis that FRa negatively regulates miRNAs associated with Oct4 and its downstream target effector Trim71, we first ascertained whether FA treatment decreased miR-138 and miR-let-7 levels. Cranial NT (from wild type) and open cranial neural fold (from E10.5 excencephalic Sp-/embryos) explants were prepared as described by Ichi et al. [2, 3] and grown in the absence or presence of 100 ng/ml FA for 24 hours. Subsequently, RNA was isolated from migrating NCCs and miR-let-7 and miR-138 levels were determined by qPCR (as per manufacturer’s instructions). FA significantly decreased miR-let7 and miR-138 levels (Fig. 4A). To ascertain whether changes in miRNA levels were mediated by FA/FRa, scr-shRNA or FRa-shRNA was transfected into cranial NCCs prior to treatment with FA. In the presence of scr-shRNA, FA caused a decline in miRNA levels (Fig. 4B, left panel), whereas in the presence of FRa-shRNA (Fig. 4B, right panel), miR-138 and miR-let-7 levels did not change, indicating that FA/FRa negatively regulate miRNAs that target Oct4 and Trim71. To establish if decreased levels of miR-138 and miR-let-7 affects their respective downstream targets, Oct4 and Trim71, 30 UTR-Oct4-renilla luciferase (Fig. 4C left panel) and 30 UTRTrim71-renilla luciferase (Fig. 4C right panel) reporter constructs (GeneCopoeia Inc.) were transfected into CNCCs prior to treatment with FA (12 hours) in a 48 well format, as per manufacturer’s instructions. Empty control vector (GeneCopoeia Inc.) was used as a negative control. Luciferase activity was low in the absence of FA and increased significantly in response to FA. This finding supports the hypothesis that decreased levels of miR-138 and miR-let-7, in response to FA, affect expression of their downstream targets. Decreased miR-138 and miR-let-7 and subsequent increased 30 UTR-Oct4- and 30 UTR-TRIM71-renilla luciferase activities were mediated by FRa. This was shown by cotransfection of FRa-shRNA with 30 UTR-Oct4-renilla luciferase or 30 UTR-Trim71-renilla luciferase constructs into CNCCs. Also noteworthy is that FRa-shRNA transfection blocked the decrease of miR-let-7 and miR-138 by FA.

FRa Interacts with Drosha and Pasha (DGCR8) Within the pre-miRNA Processing Complex FA via FRa negatively regulates miRNAs as seen by regulation of miR-138 and miR-let-7. Biogenesis of miRNA is regulated at multiple levels, including during miRNA transcription and processing by Drosha and Dicer in the nucleus and cytoplasm respectively. As the above results demonstrated that mature miRNAs were reduced by FA, we hypothesized that FRa could bind to Drosha or Dicer to regulate miRNAs. Coimmunoprecipitation assays were done with anti-FRa antibody, and immuno blotted (IB) with antibodies against Dicer, Drosha or DGCR8. FRa did not bind Dicer (data not shown). FRa did bind to the Drosha/DGCR8 complex at 15 min of FA treatment (Fig. 5), suggesting that FRa affects pre-miRNA processing, as opposed to miRNA maturation which is Dicer dependent. It is interesting to note that FRa-DGCR8-Drosha complex come together at 15 minutes and not earlier or later than 15 minutes of FA treatment. How FRa binding to the pre-miRNA processing complex decreases its activity remains to be determined and shall be addressed in future work.

Silencing miR-138 and miR-let-7f Restored Proliferation in Sp-/- Embryo NCCs and Resulted in Formation of “Spheres” As reported previously [2] and re-validated in Figure 4A, NTs from Sp-/- embryos show increased expression of miR-138 and miR-let-7 and decreased proliferation potential. FA supplementation to Sp1/- females prior to pregnancy decreased the expression of these miRNAs and restored proliferation potential of E10 Sp-/- neural stem cells [2]. To provide further support for the hypothesis that reduction of miR-138 and miRlet-7 in cranial NCCs is necessary and sufficient to restore proliferation in cranial neural fold explants grown from E10.5 wild type and Sp-/- excencephalic embryos, anti-miR-138 and anti-let-7 (anti-miR-let-7f2-3p and anti-miR-let-7f-5p) or scrambled control anti-miRNA (scr-anti-miRNA), were transfected into cranial NCCs migrating from wild type as well as Sp-/- cranial neural tube explants (as shown in Fig. 6A and 6B). Twenty-four hours post-transfection, the cells were moved to ultra-low attachment polystyrene six well plates in NeuroBasal plus medium supplemented with bFGF and EGF to assay proliferation, as measured by formation of spheres [2]. At 48 hours post transfection from another parallel transfection, total RNA was isolated and q-RT-PCR was performed to ascertain that anti-miR138 and anti-miR-let7-3p and antimiR-let7-5p successfully knocks down miR-138 and miR-let7 genes respectively. The results showed that anti-miR-let7-3p and anti-let-7-5p and anti-miR-138 knocked down the levels of miRNAs (Supporting Information Fig. S1). As compared with wild type cranial NCC, the Sp-/- cranial NCCs did not proliferate with scrambled anti-miR. However, transfection of anti-miR-138 or anti-miR-let-7 (anti-miR-let-7f2-3p and antimiR-let-7f-5p) restored the lost proliferative potential of Sp-/cranial NCCs (Fig. 6A, 6B, 6C). Transfection of combined antimiRNAs did not augment proliferation (data not shown).


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Figure 6. Silencing miR-138 and miR-let-7 restored proliferation in Sp-/- cranial NCCs and resulted in the formation of spheres. E10.5 wild type (A) and Sp-/- (B) mouse embryo cranial NT or open neural fold explants were grown in matrigel coated six well plates in NeuroBasal medium supplemented with bFGF and EGF. After 7 days the explants were carefully removed and pre-migratory NCCs which had adhered to matrigel were subjected to papain for 5 minutes and re-plated in fresh matrigel coated six well plates. After 24 hours, cells were transfected with anti-scrambled miR (negative control) anti-miR-138, anti-miR-let-7f2-3p or anti-let-7f-5p. Twenty-four hours post transfection, the cells were lifted by trypsin and allowed to grow in suspension in ultra-low attachment six well plates. Cells transfected with anti-miR-138, anti-miR-let-7f2-3p or anti-let-7f-5p showed sphere formation both in wild type and Sp-/-; (C): Quantification of the results obtained in (A) and (B). (D): Knockdown of Oct4 and Trim71 abolishes the FA/FAa/miR-138/miR-let-7f-mediated restoration of proliferation of Sp-/- cranial neural crest cells. n 5 9 and each data point in triplicate. *, p < .05; ***, p < .0001; (E): Oct4 and Trim71 expression level is higher in the cells with miR-138 and miR-let-7f-3p and miR-let-7f-5p knockdown than in control cells (ScrAnti-miR). n 5 3 and western blots are representative western blots. Abbreviations: NCCs, neural crest cells.

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Figure 7. Hypothetical model of FRa function in NCCs. In response to FA, FRa translocates to the nucleus: (a) where it binds the cis-regulatory elements of Oct4, Sox2, and Klf4, activating transcription; (b) this in turn activates downstream targets such as Trim71; and (c) downregulates levels of miR-138 and miR-let-7, which target Oct4 and Trim71 respectively; (d) thus removing the inhibitory effects on Oct4 and Trim71 expression. Abbreviations: FA, folic acid; FRa, folate receptor alpha; NCCs, neual crest cells.

Taken together, these results indicate that reduction of miR138 or miR-let-7 is sufficient for rescuing and maintaining the proliferation potential of cranial NCCs in FA-mediated rescue of Sp-/- embryos. To test whether knockdown of Oct4 and Trim71 can abolish the FA/FAa/miR-138/miR-let-7f-mediated restoration of proliferation of Sp-/- CNCCs, FA rescued Sp-/- CNCCs were transfected with Sh-Oct4 and Sh-Trim71 and the colony forming units counted after 7 days of transfection. The results showed that knocking down Oct4 and Trim71 in FA rescued Sp-/- CNCCs abolished their proliferation potential (Fig. 6D). To confirm whether the Oct4 expression level is higher in the cells with miR-138 and miR-let-7 knockdown than in control cells (anti-scr-miR), q-RT-PCR of Oct4 and Trim71 as well as Oct4 and Trim71 immunoblots of the Sp-/- CNCCs transfected with anti-miR-138, anti-miR-let-7-3p, anti-miR-let7-5p and anti-scr-miR control were performed. The results showed that Oct4 expression (RNA and protein) was higher in anti-miR-138 transfected cells although not very significant (Fig. 6E). Similarly, Trim71 expression was higher in anti-miR-let-7-3p than with anti-miR-let-7-5p transfection (Fig. 6F). These observations go toward proving the concept of the proposed mechanisms of FA/FRa/miR-138/Oct4 and FA/FRa/miR-138/Trim71 in FA mediated restoration of proliferation of Sp-/- CNCCs.

DISCUSSION Role of Oct4 in maintaining and regaining stem cell pluripotency is well documented [28, 29]. Of particular interest is that FRa bound Oct4 at both enhancer and promoter regions whereas binding with Sox2 and Klf4 were limited to promoter regions. Our data shows that at 12 hours, both Oct4 and Trim71 were upregulated by FA, whereas there was a gradual and time dependent increase in Klf4 and Sox2 in O9-1 cells. It is possible that FA/FRa first increases the Oct4 expression, which in turn upregulates its downstream target Trim71. Since the pluripotency genes and microRNAs have been reported to exist as a transcriptional regulatory network [30, 31], it is plausible that FRa mediated downregulation of miRNAs tar-


geting Klf4 and Sox2 takes some time for upregulating these genes in these CNCC line. The Oct4 enhancer interactome is enriched in transcription factor binding sites [32], which may mediate chromatin interactions governing NCSC fate. Oct4 gene expression is governed by upstream distal and proximal enhancers [31]. A set of 13 sequence-specific transcription factors (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12) interact with the Oct4 enhancer [33]. Additionally, the Oct4 distal enhancer, which achieves regulation by spatial proximity via looping [34, 35], contains active histone marks (H3K4me1, H3K27ac, H3K4me3, and H3K9ac), and active cis regulatory sequences (DNA hypersensitivity sites), 5hydroxymethylcytosine (5-hmc), in addition to early DNA replication domains [32]. Genes such as Oct4 are associated with active enhancers known as super-enhancers [36]. FRa binding to the Oct4 enhancer, while simultaneously binding promoters of three master regulators of pluripotency, demonstrates a molecular mechanism linking FA to regulation of NCSC proliferation. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 in neural crest cells argues against a transient pluripotent state during reprogramming [37], but FA/FRa induced increase in the expression of pluripotency genes could be a new way to derive pluripotent stem cells from NCSC. Our results also show that FRa downregulates the levels of miR-138 and miR-let-7 which target Oct4 and its downstream effector Trim71, respectively. The let-7 family of miRNAs is not expressed in pluripotent cells [17]. However, they are abundantly expressed in somatic cells and promote terminal differentiation during development [17]. The biogenesis of miR-let-7 during mouse embryogenesis is inhibited by a stem cell renewal factor called LIN28, an RNA binding protein [38]. In mouse embryos, Trim71 is expressed in epiblasts and embryonic ectoderm by E9.5 with an expression pattern that is opposite to that of let-7 [39]. Trim71 and let-7 functionally repress each other in a negative feedback loop [21]. Trim71 knockout mouse embryos show developmental abnormalities at E9.5 and die as a result of NT closure defects [40]. Mitschka et al. [41] showed that in mouse embryonic stem cells, Trim71 is dispensable for self-renewal but its loss results in up-regulation of genes required for neural development, suggesting that Trim71 is associated with the priming of neural differentiation rather than with pluripotency. Thus, the activation of the LIN28/let- 7/Trim71 axis is important for early development, whereas its repression is crucial for successful reprogramming [42]. FRa mediated down-regulation of miR-let-7 thereby resulting in an up-regulation of Trim71 is similar to the action of LIN28, in that both FRa and LIN28 inhibit the biogenesis of miR-let-7. Our observation of FRa binding to the distal Oct4 enhancer suggests a role for FRa in the integration of external FA signal with the core transcriptional network in NCCs. Alternatively, FRa could be yet another stem cell renewal transcription factor similar to LIN28 [38]. The super-enhancers with FRa binding likely act to control genes important for NCC type specification. Indeed, the role of FRa in the epigenetic control of super-enhancer activity in NCCs is critical to our understanding of normal


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developmental processes, and will require additional investigation with further molecular specificity. In summary, the hypothetical model of FRa function in CNCCs is depicted in Figure 7. In response to FA, FRa translocates to the nucleus where it binds the cis-regulatory elements of Oct4, Sox2, and Klf4, activating their transcription. This in turn activates downstream targets such as Trim71 (a downstream target of Oct4) and downregulates levels of miR138 and miR-let-7, which target Oct4 and Trim71 respectively. Thus, the results presented here suggest a novel pleiotropic role of FRa: (a) direct activation of genes associated with maintaining stem cell characteristics, in this case Oct4, Sox2, and Klf4 genes; and (b) repression of miRNA biogenesis associated with these genes or their effector molecules, as demonstrated for Oct4 and its effector target Trim71. It must be noted that this model is far from complete because it focuses only on the FRa regulation of miR-138/Oct4/Trim71 and miRlet-7/Trim71 axis and does not take into account the FRa mediated down-regulation of miR-337-3p and miR-148a that target Sox2 and Klf4 respectively [2]. Future work shall test this hypothesis.

CONCLUSION

AND

SUMMARY

This work identifies two novel roles of FRa: (a) Transcriptional regulation of Oct4, Sox2, and Klf4 in CNCCs through binding at cis-regulatory elements; and (b) negative regulation of miRNAs, such as miR-138 (which targets Oct4) and miR-let-7 (which targets Trim71, a downstream effector target of Oct4). This is critical because these functions of FRa at an early developmental time point provide an explanation as to why FA supplementation prior to pregnancy obviates NTDs. Moreover, FRa regulation of the miR-138/Oct4/Trim71 and miR-let7/Trim71 axis may help explain how pre-migratory NCCs maintain their multipotent phenotype and proliferation potential prior to differentiation.

REFERENCES 1 Nakazaki H, Reddy AC, Mania-Farnell BL et al. Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development. Dev Biol 2008;316:510–523. 2 Ichi S, Costa FF, Bischof JM et al. Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development. J Biol Chem 2010;285:36922– 36932. 3 Ichi S, Nakazaki H, Boshnjaku V et al. Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid. Stem Cells Dev 2012;21:321–330. 4 Ratnam M, Marquardt H, Duhring JL et al. Homologous membrane folate binding proteins in human placenta: Cloning and sequence of a cDNA. Biochemistry 1989;28: 8249–8254. 5 Spiegelstein O, Cabrera RM, Bozinov D et al. Folate-regulated changes in gene expression in the anterior neural tube of folate binding protein-1 (Folbp1)-deficient

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ACKNOWLEDGMENTS Thanks are due to Dr. Mary J. Hendrix for critically reviewing the manuscripts and giving valuable suggestions during the course of the study. This work was supported by the State of Illinois Excellence in Academic Medicine award (CSM), a Grant from the Spastic Paralysis Research Foundation of IllinoisEastern Iowa District of Kiwanis (CSM. and DGM); the Spina Bifida Association and CHCRC Pilot Grant award (CSM); Eleanor Clarke Chair in Developmental Neurobiology (CSM); Francis Chou from Chou Mutual Funds for providing financial support for research in Neural Tube Defects. We thank BioRad for their generous gift of Trans-Blot Turbo and the ChemiDocMP system. piSicoR-mCh-Oct4i was a generous gift from Miguel Ramalho-Santos (Addgene plasmid # 21906).

AUTHOR CONTRIBUTIONS V.M., A.S., S.M.: conception and design; collection and/or assembly of data; final approval of manuscript; E.A.: collection and/or assembly of data; final approval of manuscript; M.R.S.: conception and design; collection and/or assembly of data; data analysis and interpretation; final approval of manuscript; S.I., T.T.: conception and design; final approval of manuscript; B.M.-F.: conception and design; manuscript revision/editing; final approval of manuscript; D.G.M.: conception and design; data analysis and interpretation; final approval of manuscript; C.S.M.: conception and design; collection and/or assembly of data; data analysis and interpretation; manuscript writing; final approval of manuscript. V.M. and A.S. contribution equally to this article.

DISCLOSURE

OF

POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

murine embryos. Neurochem Res 2004;29: 1105–1112. 6 Tang LS, Finnell RH. Neural and orofacial defects in Folp1 knockout mice [corrected]. Birth Defects Res Part A Clin Mol Teratol 2003;67:209–218. 7 Tang LS, Santillano DR, Wlodarczyk BJ et al. Role of Folbp1 in the regional regulation of apoptosis and cell proliferation in the developing neural tube and craniofacies. Am J Med Genet Part C Semin Med Genet 2005; 135c:48–58. 8 Tang LS, Wlodarczyk BJ, Santillano DR et al. Developmental consequences of abnormal folate transport during murine heart morphogenesis. Birth Defects Res Part A Clin Mol Teratol 2004;70:449–458. 9 Wallingford JB, Niswander LA, Shaw GM et al. The continuing challenge of understanding, preventing, and treating neural tube defects. Science 2013;339: 1222002. 10 Rosenquist TH, Chaudoin T, Finnell RH et al. High-affinity folate receptor in cardiac neural crest migration: A gene knockdown model using siRNA. Dev Dyn 2010;239:1136– 1144.

11 Boshnjaku V, Shim KW, Tsurubuchi T et al. Nuclear localization of folate receptor alpha: A new role as a transcription factor. Sci Rep 2012;2:980. 12 Kaltschmidt B, Kaltschmidt C, Widera D. Adult craniofacial stem cells: Sources and relation to the neural crest. Stem Cell Rev 2012;8:658–671. 13 Achilleos A, Trainor PA. Neural crest stem cells: Discovery, properties and potential for therapy. Cell Res 2012;22:288–304. 14 Eberhart JK, He X, Swartz ME et al. MicroRNA Mirn140 modulates pdgf signaling during palatogenesis. Nat. Genet 2008;40: 290–298. 15 Cordes KR, Srivastava D. MicroRNA regulation of cardiovascular development. Circ Res 2009;104:724–732. 16 Subramanyam D, Blelloch R. From microRNAs to targets: Pathway discovery in cell fate transitions. Curr Opin Genet Dev 2011;21:498–503. 17 Ambros V. MicroRNAs and developmental timing. Curr Opin Genet Dev 2011;21: 511–517.



12

18 Li Z, Rana TM. Molecular mechanisms of RNA-triggered gene silencing machineries. Acc Chem Res 2012;45:1122–1131. 19 Pasquinelli AE. MicroRNAs and their targets: Recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 2012;13:271–282. 20 Zehir A, Hua LL, Maska EL et al. Dicer is required for survival of differentiating neural crest cells. Dev Biol 2010;340:459– 467. 21 Worringer KA, Rand TA, Hayashi Y et al. The let-7/LIN-41 pathway regulates reprogramming to human induced pluripotent stem cells by controlling expression of prodifferentiation genes. Cell Stem Cell 2014;14: 40–52. 22 Belteki G, Haigh J, Kabacs N et al. Conditional and inducible transgene expression in mice through the combinatorial use of Cremediated recombination and tetracycline induction. Nucleic Acids Res 2005;33:e51. 23 Ishii M, Arias AC, Liu L et al. A stable cranial neural crest cell line from mouse. Stem Cells Dev 2012;21:3069–3080. 24 Ishii M, Han J, Yen HY et al. Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest. Development (Cambridge, England) 2005;132:4937–4950. 25 Jiang X, Rowitch DH, Soriano P et al. Fate of the mammalian cardiac neural crest. Development (Cambridge, England) 2000; 127:1607–1616.

26 Mayanil CS, George D, Freilich L et al. Microarray analysis detects novel Pax3 downstream target genes. J Biol Chem 2001;276: 49299–49309. 27 Mayanil CS, Pool A, Nakazaki H et al. Regulation of murine TGFbeta2 by Pax3 during early embryonic development. J Biol Chem 2006;281:24544–24552. 28 Shi G, Jin Y. Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res Ther 2010;1:39. 29 Esch D, Vahokoski J, Groves MR et al. A unique Oct4 interface is crucial for reprogramming to pluripotency. Nat Cell Biol 2013; 15:295–301. 30 Kim J, Chu J, Shen X et al. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 2008;132:1049–1061. 31 Young RA. Control of the embryonic stem cell state. Cell 2011;144:940–954. 32 Cai M, Gao F, Zhang P et al. Analysis of a transgenic Oct4 enhancer reveals high fidelity long-range chromosomal interactions. Sci Rep 2015;5:14558. 33 Chen X, Xu H, Yuan P et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 2008;133:1106–1117. 34 Gao F, Wei Z, Lu W et al. Comparative analysis of 4C-seq data generated from enzyme-based and sonication-based methods. BMC Genomics 2013;14:345.

35 Meshorer E, Misteli T. Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol 2006;7:540–546. 36 Whyte WA, Orlando DA, Hnisz D et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 2013;153:307–319. 37 Weber M, Apostolova G, Widera D et al. Alternative generation of CNS neural stem cells and PNS derivatives from neural crestderived peripheral stem cells. Stem Cells (Dayton, Ohio) 2015;33:574–588. 38 Viswanathan SR, Daley GQ. Lin28: A microRNA regulator with a macro role. Cell 2010;140:445–449. 39 Rybak A, Fuchs H, Hadian K et al. The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nat Cell Biol 2009;11: 1411–1420. 40 Maller Schulman BR, Liang X, Stahlhut C et al. The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle (Georgetown, Tex) 2008;7:3935– 3942. 41 Mitschka S, Ulas T, Goller T et al. Coexistence of intact stemness and priming of neural differentiation programs in mES cells lacking Trim71. Sci Rep 2015;5:11126. 42 Takahashi K, Yamanaka S. A developmental framework for induced pluripotency. Development (Cambridge, England) 2015; 142:3274–3285.

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