|Journal of Cancer Stem Cell Research (2017), 5:e1001
© 2017 Creative Commons. All rights reserved ISSN 2329-5872
|Research Article||Open Access|
|The Transcription Factor CP2-like 1 Is Expressed in Very Small Embryonic-like Stem Cells and Other Adult Stem Cells: Implications for Cancer Stem Cells|
|Hye-Yeon Lee1,2,§, Hyein Ju1,2,§, Jinbeom Heo1,2, YongHwan Kim1,2, Jisun Lim1,2, Seungun Lee1,2, Hwan Yeul Yu1,2, Chae-Min Ryu1,2, Ju-Young Han1,2, Sabine J. Waigel3, Yinlu Chen4 and Dong-Myung Shin1,2,*|
|1Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea.|
|2Department of Physiology, University of Ulsan College of Medicine, Seoul, 05505, Korea.|
|3Genomics Facility and Department of Medicine, University of Louisville, KY 40202, USA.|
|4Genomics Facility and Department of Anatomical Sciences and Neurobiology, University of Louisville, KY, 40202, USA.|
|§These authors equally contributed to this work.|
|*Corresponding Author: Dong-Myung Shin, Ph.D., Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Pungnap-2 dong, Songpa-gu, Seoul, 05505, Korea. Tel: 82-2-3010-2086; Fax: 82-2-3010-8493; E-mail: email@example.com|
Received: March 5, 2017; Revised: March 28, 2017;
Accepted: March 28, 2017
Abstract: A population of very small embryonic-like stem cells (VSELs) in adult tissues, similar to embryonic stem cells (ESCs), is capable of differentiation in in vitro conditions and in vivo animal models into cells of all three germ lineages. The open chromatin structure of pluripotency genes and genomic imprinting-related epigenetic mechanisms maintain their pluripotent and quiescent state, respectively. However, which transcription factors (TFs) are commonly expressed to maintain pluripotency in these SCs remains unknown. Here, by comparing the global transcriptome of VSELs with that of adult stem cells (SCs) [e.g., hematopoietic SC (HSCs)] or ESCs, we demonstrated that transcription factor CP2-like 1 (Tfcp2l1l), a well-known naïve factor for ESCs, is highly expressed in murine VSELs. By analyzing a single-cell-level transcriptome database established from highly purified murine bone marrow (BM)-derived VSELs and HSCs as well as ESCs, we found that expression in a subset of TFs was shared by VSELs and ESCs. Among them, Tfcp2l1 was commonly expressed in murine VSELs and ESCs but not in HSCs and terminally differentiated BM mononuclear cells. During the differentiation of ESCs by forming an embryoid body or by treatment with retinoic acid, the expression of Tfcp2l1 decreased more rapidly than that of the typical pluripotency-associated TFs, including Oct4, Nanog, and Sox2. Ectopic expression of Tfcp2l1 in ESCs enforced expression of Oct4. Taken together, these results suggest that Tfcp2l1 functions as a common TF to regulate pluripotency in Oct4-expressing embryonic and adult pluripotent SCs and that dys-regulation of Tfcp2l1 in adult SCs could initiate the transformation into cancer stem cells.
Keywords: Tfcp2l1, Transcription factors, Pluripotency, Oct4, VSELs
Embryonic development and subsequent rejuvenation of adult tissues are regulated by a population of stem cells (SCs) that undergo self-renewal, maintain their own pool, and give rise to differentiated progenitors that replace cells used up during life . Thus, SCs are guardians of tissue/organ integrity and regulate the life span of an adult organism. The most important SC population from a regenerative point of view is pluripotent SCs (PSCs) [2, 3]. According to the definition, PSCs have to fulfill certain in vitro as well as in vivo criteria, such as (i) giving rise to cells from all three germ layers; (ii) completing blastocyst development; and (iii) forming teratomas after inoculation into experimental animals. PSCs from the inner mass of blastocysts could be expanded ex vivo as immortalized embryonic SCs (ESCs) [4, 5], which are most well-studied PSCs. Recently, PSCs could be established by somatic cell reprograming, including the transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc) [6, 7] and somatic cell nuclear transfer .
To maintain this unique pluripotent property, PSCs commonly express pluripotency core transcription factors (TFs) such as Oct4, Nanog, and Sox2 , which are turned off in differentiated somatic cells. These TFs form the pluripotent core circuitry by reinforcing the expression of genes, which are involved in keeping PSCs in an undifferentiated status but repressing differentiation-inducing transcription. Moreover, several genes that are frequently upregulated in tumors, such as Stat3 [10, 11], E-Ras , cMyc , Klf4 , and β-catenin [15, 16], have been shown to contribute to the long-term maintenance of the ES cell phenotype and the rapid proliferation of ESCs in culture.
Recently, a population of very small embryonic-like SCs (VSELs) has been identified in murine adult tissues, including the bone marrow (BM), fetal liver, testes, ovaries, and human umbilical cord blood [17–21]. VSELs are smaller than erythrocytes and express several markers of (i) pluripotency (Oct4, Nanog, Sox2, and SSEA-1), (ii) epiblasts (Gbx2, Fgf5, and Nodal), and (iii) epiblast-derived migratory primordial germ cells (PGCs) (Stella, Blimp1, and Prdm14) [22, 23]. The true expression of Oct4, Nanog, and Stella in murine BM-derived VSELs has been confirmed by demonstrating the demethylated state of DNA and enrichment of transcriptionally active histone codes in the promoters of these genes [22, 24]. Furthermore, epigenetic changes in the expression of some imprinted genes that are paternally (Igf2-H19 and RasGRF1) and maternally methylated/imprinted (Igf2R and KCNQ1) maintain the quiescence of VSELs . VSELs can differentiate into cells from all three germ layers in in vitro culture conditions. Using several in vivo tissue regeneration animal models, VSELs can be specified in vivo into mesenchymal SCs (MSCs) , cardiomyocytes , type II alveolar cells , and long-term engrafting hematopoietic SCs (HSCs) [28, 29]. In normal physiological condition, proliferation and developmental potency of VSELs should be tightly modulated by a unique epigenetic reprogramming in genomic imprints to protect VSELs from uncontrolled proliferation and teratoma formation. In pathological situations, however, unleashed VSELs could contribute to the development of several malignancies . However, the precise molecular mechanism by which primitive VSELs control their pluripotency, proliferation and differentiation potential remains to be determined.
As mentioned, TFs, including Oct4, Sox2, Klf4, Nanog, and Stat3, and chromatin regulatory proteins play a major role in the regulation of self-renewal by maintaining an ESC-specific gene expression pattern . However, the precise profile of these pluripotency-associated TFs in Oct4-expressing adult VSELs is not well understood. In this regard, we examined TFs that would show common expression patterns in VSELs and ESCs using single-cell-level transcriptome databases from several types of PSCs and adult cells . In this present study, we demonstrated that TF CP2-like 1 (Tfcp21l), a well-known naïve factor for ESCs, was highly expressed in murine BM-derived VSELs.
We employed a microarray database representing a cDNA library established from 20 cells of FACS-sorted VSELs, HSCs, or trypsinized ESC-D3 cells. All the procedures for 20-cell cDNA library synthesis, microarrays, and data processing have been described in our previous report . The microarray datasets discussed in the present study have been deposited in NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE29281. Heatmap analyses with hierarchical clustering of microarray data were performed using Partek software (Partek Inc, Saint Louis, MO), and gene network functional analysis was performed using Ingenuity pathway analysis (IPA) software version 8.7 (Ingenuity Systems, Inc. Redwood, CA) by core and comparison analysis for gene networks, bio-functions, and canonical pathways. Hierarchical agglomerative clustering with Spearman's rank correlation coefficient and average linkage were applied to both rows (samples) and columns (probe sets), and heatmaps were produced by arranging the rows and columns according to the clustering outputs. Gene network analysis was performed using IPA software by default setting. The minimum resolution for multiple probes was set at the experimental p value. Red and green represented upregulated and downregulated values, respectively. The bio-function and canonical pathway analysis of the indicated gene lists was performed using the default settings, with a threshold value of 0.05 and Fisher's exact test for scoring method.
Total RNA from various cells was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA), including treatment with DNase I (QIAGEN). mRNA (400 ng) was reverse-transcribed with Taqman Reverse Transcription Reagent (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. The resulting cDNA fragments were amplified using Amplitaq Gold at one cycle of 8 min at 95°C; two cycles of 2 min at 95°C, 1 min at 62°C, and 1 min at 72°C; 35 subsequent cycles of 30 s at 95°C, 1 min at 62°C, and 1 min at 72°C; and one cycle of 10 min at 72°C. All primers were designed with Primer Express software (Applied Biosystems) as at least one primer included an exon and intron boundary. They are available upon request.
For quantification of the expression of the indicated transcripts, cDNA templates, prepared using both regular total RNA-reverse transcription and single-cell-level protocols, were amplified with SYBR Green PCR Master Mix (Applied Biosystems) using RQ-PCR on the PikoReal™ Real-Time PCR System (Thermo Fisher Scientific, Pittsburgh, PA). All primers were designed with Primer Express software (Applied Biosystems) as at least one primer included an exon–intron boundary. The threshold cycle (Ct), the cycle number at which the fluorescence of the amplified gene reaches a fixed threshold, was subsequently determined, and relative quantification of the expression level of target genes was performed through the 2−ΔΔCt method using the mRNA level of Gapdh as an endogenous control gene and that of the indicated cells as a calibrator.
ESCs (R1 line) were grown in DMEM-high glucose medium (HyClone, Pittsburgh, PA) supplemented with 2 mM L-glutamine, 20 mM HEPES, MEM nonessential amino-acid, penicillin/streptomycin solution (Cellgro, Pittsburgh, PA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St Louis, MO), 15% heat-inactivated FBS (Hyclone), and 100 IU/ml ESGRO (Millipore, Billerica, MA) in gelatin-coated tissue culture dishes as previously described . Embryoid body (EB) formation was performed using the hanging drop method in Petri dishes. In total, 1 μM retionic acid (Sigma-Aldrich) was used to induce the differentiation of ESCs. The undifferentiated status of ESCs was assessed using the Alkaline Phosphatase (AP) Detection Kit (Millipore), according to the manufacturer's instructions.
The open reading frame of murine Tfcp2l1 was directly amplified from a murine ES cell cDNA library with following the primers: mTfcp2l1_ORF_F: GGAT-CCGCCACCATG-CTGTTCTGGCACACGCAG and mTfcp2l1_ORF_R: CTCGAGTCAGAGTCCACACTT-CAGGAT. The amplified Tfcp2l1 ORF was cloned into pCMV_3Tag-1 vector for overexpression of the Flag-tagged Tfcp2l1 protein. The plasmid containing the Tfcp2l1 construct was transfected into murine ESCs using Lipofectamine 2000 (Life Technologies, La Jolla, CA). To examine the expression level of the indicated proteins, cell extracts (30 μg) were prepared in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) and separated on 10% SDS-PAGE gels. The protein expression level was assessed by probing with monoclonal antibodies specific to Oct4 (Santa Cruz Biotechnology), Tfcp2l1 (Aviva Systems Biology, San Diego, CA), Sox2 (Epitomics, Burlingame, CA), Nanog (Abcam, Cambridge, UK), Tet1 (Millipore), and Flag epitope (Sigma-Aldrich). Relative protein expression was calculated by normalization to β-actin (Santa Cruz Biotechnology).
For immunocytochemistry, ESCs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 24 h and co-stained using anti-Oct4 mouse IgG monoclonal antibody (Millipore) and anti-Tfcp2l1 rabbit IgG polyclonal antibody. Immunostaining was visualized using Alexa 488- or 564- conjugated anti-mouse or anti-rabbit antibodies (Molecular Probes, Grand Island, NY). The nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI, Sigma-Aldrich). The stained samples were photographed using an inverted fluorescence microscope (EVOS® FL Color Imaging System, Life Technologies).
All data were analyzed by one- or two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) was used to perform all analyses. Statistical significance was defined as p < 0.05 or p < 0.01.
VSELs share several molecular natures with ESCs to maintain pluripotency. Because TF networks determine the expression of genes involved in many biological processes , we attempted to identify the TFs genes shared by VSELs and ESCs. To address this issue, we re-analyzed previously published data (GSE29281), consisting of a transcriptome of 20 FACS-sorted cells from murine BM-derived VSELs, HSCs, and the ESC line ESC-D3 . By setting the cutoff as two fold upregulated or downregulated genes simultaneously in VSEL and HSC as well as ESC and HSC comparison, with a statistical p-value cutoff of 0.000491342 (FDR = 0.01 in VSEL and HSC comparison), we found 589 upregulated and 1553 downregulated genes (Figure 1A). By further filtering the selected genes using the ontology term “Transcription” we found that 129 upregulated and 262 downregulated genes were enriched as pluripotency-related genes. Heatmap clustering analysis using these selected genes demonstrated that transcriptomes of both PSCs (VSELs and ESCs) were closely clustered with each other but distant from those of differentiated adult SCs (HSCs) (Figure 1B), confirming that this selected gene set properly represents the TF profile characteristic of PSCs.
Figure 1. Gene set characterizing TFs shared by murine VSELs and ESCs. (A) Summary of the analysis of microarray data, focusing on pluripotency-associated transcription factors (TFs). FDR; false discovery rate, GO; gene ontology, VSEL; very small embryonic-like stem cell, HSC; hematopoietic stem cell, ESC; embryonic stem cell. (B) Heatmap analysis with hierarchical clustering for genes related to TFs and enriched in both PSCs (VSELs and ESCs). Blue, red, and green bars represent HSCs, ESCs, and VSELs respectively. (C) Gene network analysis of Tfcp2l1. Gene networks are illustrated by overlaying all experimental values for the ESC and HSC comparison datasets. Upregulated and downregulated genes in the heatmap and gene network analyses are represented as red and green colors, respectively.
Next, we performed gene network analysis, which is an all-comprehensive approach for identifying the molecules interacting with target genes with respect to all biological events, including transcription regulation, upstream or downstream regulation, and protein–protein interaction . To focus on genes involved in the regulation of pluripotency, putative targets were selected if their gene network included more than two interacting pluripotency-related genes (e.g., Oct4, Nanog, Sox2, Rex1, Sall4, and Stat3) (Figure 1C). Accordingly, we found four putative target genes, including Tfcp2l1, PR domain-containing protein 5 (Prdm5), WD repeat-containing protein 77 (Wdr77), and CBP/p300-interacting transactivator with glu/asp-rich c-terminal domain 1 (Cited1).
By employing the RQ-PCR assay, we confirmed that Tfcp2l1 transcripts, similar to Oct4 and Nanog ones, were specifically found in VSELs and ESCs, but few were found in MEF and other adult tissue cells (HSCs and BMMNCs) (Figure 2A). The expression of Prdm5 was detected in embryonic tissue cells and only in VSELs in adult tissue cells (Figure 2A and Figure 2B). In contrast, the expression of Wdr77 and Cited1 was the highest in HSCs and BMMSCs, respectively, but they were hardly detected in either VSELs or ESCs. Among the validated genes, Tfcp2l1 showed the highest enrichment in the expression dataset (16.24-fold increase in VSELs vs. HSCs) and gene network analysis; it has recently been reported as the missing pluripotency-associated TF in both murine [33, 34] and human ESCs . Indeed, the immunofluorescent staining of undifferentiated ESC colonies demonstrated that Tfcp2l1 located in the nucleus clearly co-localized with Oct4 (Figure 2C).
Figure 2. Expression of Tfcp2l1 in murine VSELs and ESCs. (A and B) RQ-PCR (A) and RT-PCR (B) of pluripotency-associated TFs in the indicated stem cells (SCs) or bone-marrow mononuclear cells (BMMSCs). The relative expression level of genes is represented as the ratio of the indicated stem cells value to that obtained for ESCs and is shown as the mean ± SEM; n = 4, ***p < 0.001, compared with HSCs, two-way ANOVA with Bonferroni post-hoc tests. RT-PCR showed consistent expression of Oct4 and Tfcp2l1 in ESC and VSELs. Gapdh was used as an internal control. D.W.; distilled water. (C) Representative images of immunofluorescent staining for Oct4 (green) and Tfcp2l1 (red) proteins in murine ESCs (upper panel; ×400 magnification, scale bar = 200 μm, lower panel; ×400 magnification, scale bar = 100 μm). Nuclei were counterstained with DAPI (blue).
Next, we examined the change in the expression of Tfcp2l1 during the differentiation of ESCs. We employed two differentiation methods: EB culture using the hanging drop method and the addition of retinoic acid (RA) to the growth medium, which inactivates Oct4 transcription, which is directly mediated by trans-acting repressors such as ARP-1, COUP-TF1, and GCNF [36, 37]. During differentiation following both EB and RA treatment, the expression of Tfcp2l1 decreased similar to that of other PSC markers, including Oct4, Nanog, and Sox2 (Figure 3A and 3B). In particular, we noted that the expression of Tfcp2l1 was more rapidly repressed than that of other pluripotency-associated TFs, suggesting the significance of Tfcp2l1 in controlling pluripotency. The downregulation of Tfcp2l1 during ESC differentiation was confirmed at the protein level by western blot analysis (Figure 3C and 3D).
Figure 3. Expression of Tfcp2l1 during differentiation of murine ESCs. (A and B) ESC differentiation was induced by embryoid body (EB) formation (A) or retinoic acid (RA) treatment (B). RQ-PCR results are represented by the relative expression level as the ratio of the value of the indicated cells to that of undifferentiated ESCs and are shown as the mean ± SEM; n = 4. RT-PCR analysis of the indicated genes is shown at the bottom of each RQ-PCR result. (C and D). Western blot analysis of Tfcp2l1 during ESC differentiation induced by EB formation (C) or treatment of RA (D). The right-sized and nonspecific bands in the Nanog and Tfcp2l1 western blot data are indicated by an arrow and asterisk, respectively. β-actin was used as an internal control. W.B.; Western blot.
Next, to determine the relationship between Tfcp2l1 and Oct4, a key TF associated with pluripotency, we overexpressed Tfcp2l1 in murine ESCs. Western blot analysis confirmed that Oct4 was efficiently induced in a dose- and time-dependent manner by the ectopic expression of Tfcp2l1 (Figure 4A), suggesting that Tfcp2l1 could positively regulate the expression of Oct4. Accordingly, ESCs ectopically expressing Tfcp2l1 exhibited a stronger AP-positive staining profile than control cells (Figure 4B). Taken together, these data indicate that the expression of Tfcp2l1 positively correlates with pluripotency in embryonic and adult tissue SCs and that Tfcp2l1 plays an essential role in maintaining self-renewal and an undifferentiated state.
Figure 4. Ectopic expression of Tfcp2l1 reinforces Oct4 expression in murine ESCs. (A) Western blot analysis of ESCs transfected with the Flag-tagged Tfcp2l1 expression construct, with the indicated amount of plasmid and post-transfection periods. β-actin was used as an internal control. W.B.; Western blot. (B) Alkaline phosphatase (AP) staining of ESCs stably established with the Flag-tagged Tfcp2l1 expression construct (×200 magnification, scale bar = 200 μm). Empty: mock vector, Br: bright field.
The results presented here demonstrate that a subset of TFs is shared by murine VSELs and ESCs and that Tfcp2l1 plays a key role in maintaining a pluripotent and undifferentiated state.
Accumulating evidence has reported a population of adult tissue PSCs, which are referred to by various names, including (i) MSCs; (ii) multipotent adult progenitor cells (MAPCs); (iii) marrow-isolated adult multilineage inducible (MIAMI) cells; (iv) multipotent adult SCs (MASCs); and (v) OmniCytes [23, 38]. However, of late, there has been much debate challenging the existence of primitive PSCs in adult tissues . Thus, further investigation is required to better explain their precise molecular nature and to use them as a powerful source in regenerative medicine. In this regard, by employing genomewide gene expression databases, we identified the expression profile of TFs that could commonly characterize both ESCs and VSELs (Figure 1). Accordingly, the identified gene set included several well-known pluripotency-associated TFs, including Zfp42, Oct4, Zeb2, Nanog, Sox2, and Dnmt3l. However, we noted that this gene set was also characterized by various genes involved in basic biological processes, including cyclin E1, cell division cycle-associated 2, forkhead box O3, HIRA-interacting protein 3, and paired-like homeodomain 2, with highly ranked enrichment scores. This indicates that the transcriptional memory of PSCs in embryonic tissues may, to an undefined level, be preserved in primitive SCs deposited in adult tissues. Thus, the TF gene set shared by VSELs and ESCs identified in the present study (Figure 1) could be employed to characterize the state of pluripotency or stemness in adult tissues. This interesting possibility should be validated by further in-depth investigations. Of importance, several tumors exhibited similar morphologic and molecular features to developmentally early tissues. In addition, accumulating evidences have proven that malignancy arises from continuous serious mutation in normal stem/progenitor cells, leading to transform of them into cancer stem cells . Thus, the TF gene set shared by VSELs and ESCs could be also applicable to investigate the molecular nature of cancer stem cells found in several tumor tissues.
Among the TFs associated with pluripotency, Tfcp2l1 showed the highest enrichment scores in fold-change and gene network analysis. However, its function in adult tissues has been not well studied. Tfcp2l1 is expressed in murine and human inner cell mass of blastocysts and is downregulated shortly after implantation [41, 42]. When human ESCs were converted into a naïve-like state by the overexpression of Klf2, Klf4, and Oct4, the expression of Tfcp2l1 was found to be characteristically upregulated based on analysis of the change in the transcriptome profile . In a differentiation assay of ESCs, the expression of Tfcp2l1 rapidly decreased faster than that if other pluripotency-associated TFs (Figure 3). Furthermore, the ectopic expression of Tfcp2l1, in time- and dose-dependent manners, enforced the expression of Oct4 and AP activity in undifferentiated ESC colonies (Figure 4). These results strongly indicated that Tfcp2l1 may play a role in generating and stabilizing the murine and human pluripotent state. Thus, further studies on the mechanisms of activation and repression by Tfcp2l1 are required to resolve this. Interestingly, gene targeting of Tfcp2l1 has identified an important role of this TF in the ductal epithelium of several developing organs, including the kidney and salivary glands [44, 45], suggesting a role of Tfcp2l1 in adult tissues. In this regard, further investigation is required to determine whether the modulation of the expression or activity of Tfcp2l1 could be reliable in maintaining pluripotency or ex vivo expansion of murine or human VSELs.
Tfcp2l1 functions in gene transcription as an activator or repressor by forming DNA-binding complexes with other CP2 family members; the combination in complex components determines their transcriptional activity . This implies that these TFs should be studied as a family by carefully investigating the networks between family members expressed in a given cell type or tissue of interest. A previous study has reported that other CP2 family members such as CP2, NF2d9 and altNF2d9 are also expressed in murine ESCs . However, these CP2 family genes were not listed in the TF gene set shared by ESCs and VSELs (Figure 1B), suggesting that Tfcp2l1 plays a specific role in PSCs. Thus, further examination is required to establish whether CP2 family complexes could determine the function of this family in PSCs from embryonic and adult tissues as well as by somatic cell reprograming.
In summary, in the present study, we have identified Tfcp2l1 as a novel pluripotency-related TF shared by PSCs in embryonic (ESCs) and adult (VSELs) tissues by analyzing the transcriptomes characteristic to both types of PSCs. Tfcp2l1 plays an essential role in regulating the pluripotent state in Oct4+ embryonic or adult PSCs. Thus, the present study provides a crucial conceptional and technical advance in our understanding of not only how PSCs maintain their pluripotency but also how their transcriptional memory may be progressively sustained throughout embryonic and adult tissue development.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (grant number) (2015R1A2A1A15054754) and co-supported by the Global High-tech Biomedicine Technology Development Program of the National Research Foundation (NRF) & Korea Health Industry Development Institute (KHIDI) (MSIP&MOHW) (No. 2015M3D6A1065364). Part of this work was performed with the assistance of the University of Louisville Microarray Facility, which is supported by NCRR IDeA Awards INBRE-P20 RR016481 and COBRE-P20RR018733, the James Graham Brown Foundation, and user fees.
The authors have no competing financial interests to declare.
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