text:
Reversal of multidrug resistance by the anti-malaria drug artesunate in the esophageal cancer Eca109/ABCG2 cell line

The overexpression of ATP-binding cassette (ABC) transporters confers multidrug resistance (MDR) to tumor cells. ABCG2 is a member of the ABC superfamily. The present study aimed to investigate the correlation between ABCG2 expression and the MDR of esophageal cancer and to estimate the therapeutic benefit of downregulating ABCG2 expression and reversing chemoresistance in esophageal cells using artesunate (Art). The Eca109/ABCG2 cell line was established by transfecting the ABCG2 gene into Eca109 cells. The Eca109/ABCG2 esophageal cancer cells with ABCG2 gene overexpression were resistant to adriamycin (ADM), daunorubicin (DNR) and mitoxantrone (MIT), which indicated that ABCG2 may be associated with drug resistance in esophageal cancer. Art is a noteworthy antimalarial agent, particularly in severe and drug-resistant cancer cases, as Art is able to reverse drug resistance. In the present study, Art also exerted profound anticancer activity. The mechanism for the reversal of multidrug resistance by Art in esophageal carcinoma was analyzed using cellular experiments, but still remains largely unknown.

Introduction
Esophageal carcinoma is one of the most common malignant gastrointestinal tumors (1). The incidence of esophageal carcinoma is high in China and it is a great threat to human health. Chemotherapy is a combined modality therapy that is used in the treatment of esophageal carcinoma, however, multidrug resistance (MDR) is a major barrier to the success of chemotherapy, particularly in recurrent tumors. MDR is the resistance of cancer cells to multiple classes of chemotherapeutic drugs that may be structurally and mechanistically unrelated. When tumor cells develop drug resistance, they become resistant not only to the treated drug, but also to a variety of structurally and functionally unrelated drugs. Since the therapeutic effect is determined by this resistance, MDR has become a focus for research. There is accumulating evidence that the active export of anticancer drugs from cells is one of the major mechanisms of MDR (2,3). Tumor cells often gain drug resistance through the overexpression of membrane transport proteins that effectively efflux anticancer drugs (4,5). It has been convincingly documented that several ATP-dependent drug transporters may cause drug resistance in cancer cells by actively extruding clinically administered chemotherapeutic drugs (6). The increased transmembrane efflux of antitumor drugs is one of the best-characterized mechanisms of MDR and is mediated through the overexpression of ATP-binding cassette (ABC) transporter superfamily members. The well-known major drug transporters, ABCB1 (P-glycoprotein or MDR1) and ABCG2 (BCRP/MXR/ABCP), have been characterized in detail with respect to their structures and functions (7). These drug transporters belong to the human ABC transporter gene family that consists of at least 48 gene members. The overexpression of ABCG2 has been reported to confer drug resistance upon malignant cells to various chemotherapeutic drugs (8,9). Artesunate (Art) is a remarkable antimalarial agent, particularly in severe and drug-resistant cases (10)(11)(12). Art has also been demonstrated to exert profound anticancer activity (13)(14)(15). Art, a powerful antimalarial herbal compound, has been shown to inhibit the growth of various tumor cell lines in vitro and xenografted carcinoma in mice in vivo. Art has also been shown to inhibit the growth of esophageal cancer cells. Art may have anticancer effects on drug-resistant cells, indicating that the compound may reverse the drug resistance of cancer cells (16)(17)(18). Art has few adverse effects, so it may be developed into a drug to reverse MDR.
In the present study, the gene and protein expression of ABCG2 was detected by various experimental methods, to study the correlation between ABCG2 expression and the resistance of esophageal carcinoma. An Eca109/ABCG2 MDR cell was established by transfecting the ABCG2 gene into Eca109 cells. The ABCG2 expression level and drug efflux of the Eca109/ABCG2 cells was assessed using RT-PCR, western blot analysis and flow cytometry.
The mechanism for the reversal of multidrug resistance by Art in esophageal carcinoma was analyzed using cellular experiments. The correlation between ABCG2 expression in esophageal carcinoma and MDR, and the reversal of MDR by Art were investigated in the present study. These results may be beneficial to the chemotherapy of esophageal carcinoma in the clinic.
ABCG2 gene transfection. PCDNA3.1-ABCG2 plasmids containing ABCG2 cDNA were purchased from Jing Sai Co. (Wuhan, China). Lipofectamine 2000 (Invitrogen) was used as a transfection reagent, according to the manufacturer's instructions, and positive cell clones were selected using 600 mg/l G418 subsequent to being transfected for 72 h. The Eca109 cells that were transfected with PCDNA3.1 served as the control group. The Eca109 cells that were transfected with PCDNA3.1-ABCG2 and PCDNA3.1 were termed the Eca109/ABCG2 and Eca109/PCDNA3.1 cells, respectively. To ascertain the efficacy and specificity of the transfection, ABCG2 mRNA and protein levels were monitored using RT-PCR, western blot analysis and flow cytometry, respectively.
Cells and cell culture. The Eca109 esophageal cancer cell line was obtained from the Tumor Institute of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). The Eca109 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 5% penicillin (100 U/ml) and streptomycin (100 mg/ml) in a humidified atmosphere of 95% air and 5% CO 2 at 37˚C. The medium was changed three times a week. The Eca109/ABCG2 cells were maintained in RPMI-1640 supplemented with 10% FBS and 300 mg/l G418.
Cytotoxicit y assay. The sensitivity of the Eca109, Eca109/ABCG2 and Eca109/PCDNA3.1 cells to the anticancer drugs [adriamycin (ADM), daunorubicin (DNR) and mitoxantrone (MIT)] was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the capacity of viable cells to metabolize a yellow tetrazolium salt, MTT, using mitochondrial succinate dehydrogenase, into purple formazan crystals when dissolved in acidified propan-2-ol; the resulting purple solution is then spectrophotometrically measured at 490 nm. The cells were seeded into 96-well culture plates at a density of 5x10 4 cells/ml. The serial concentrations of the anticancer drugs, ADM, DNR and MIT, were added in a final volume of 200 µl/well. Following the drug treatment for 72 h, the medium was replaced with an equal volume of fresh medium containing 0.5 mg/ml MTT and incubated for 4 h. The medium was removed and 180 µl DMSO was added and incubated for 10 min at room temperature. The cytotoxic effects of the drugs were determined according to the optical density (OD) values using a microplate reader at an absorption wavelength of 490 nm. The cell viability is expressed as the relative formazan formation in the treated samples compared with the control cells (A490 treated cells / A490 control cells x 100%).
Flow cytometry. The level of ABCG2 protein expression, the apoptosis rate and the ADM content were analyzed in the Eca109 cells using flow cytometry.
The cells were harvested with trypsin-EDTA (1:20), washed with phosphate-buffered saline (PBS) and centrifuged for 5 min at 1,200 x g. The FITC-conjugated anti-ABCG2 antibody was added and the cells were incubated at room temperature for 30 min in the dark. The labeled cells were washed in PBS, centrifuged for 5 min at 1,200 x g and kept at 4˚C until they were used.
The cells were dyed in 1 ml fluorescence liquor containing 50 µg/ml propidium iodide (PI) following incubation for 30 min in the dark at 4˚C, then kept at 4˚C until they were used.
The ADM in the Eca109 cells exhibited red fluorescence, which was detected by a 488 nm laser and analyzed using flow cytometry.
The cells were analyzed using a Beckman Coulter Epics-XL Ⅱ type cytometer equipped with a 488 nm argon ion laser (Beckman Coulter, Miami, FL, USA). For each sample, 10,000 events selected in the living cell gate were measured. Forward scatter (FSC) and side scatter (SSC) data were used to establish a gate excluding the dead cells and debris.
ADM accumulation and efflux by flow cytometry. The cellular accumulation and efflux of ADM were analyzed by flow cytometry. The Eca109, Eca109/PCDNA3.1 and Eca109/ABCG2 cells (4x10 5 ) were incubated with 0.02 µg/ml ADM at 37˚C for 2 h and washed twice with ice-cold PBS. The cells were resuspended in ADM-free RPMI-1640 for 1 h. The cells were washed with ice-cold PBS and the ADM that was retained in the cells was detected by flow cytometry.
In the drug efflux studies, 4x10 5 Eca109/ABCG2 cells were incubated with 0.02 µg/ml ADM at 37˚C for 2 h and washed twice with ice-cold PBS. The cells were resuspended in ADM-free RPMI-1640 in the absence or presence of 1 µmol/l Art for 1 h. The cells were washed with ice-cold PBS and the ADM that was retained in the cells was detected by flow cytometry.
Total mRNA isolation and RT-PCR. Total RNA was extracted using TRIzol according to the manufacturer's instructions and then treated with AMV reverse transcriptase to form cDNA (Promega Corporation, Madison, WI, USA). The cDNA was amplified by PCR using TaqDNA polymerase, which was performed by denaturation at 94˚C for 30 sec, annealing at 57˚C for 30 sec and extension at 72˚C for 30 sec. This was performed for 35 cycles. The PCR-amplified products were run on a 1.5% agarose gel and visualized by ethidium bromide staining. The expression intensities of the optimized bands were quantified using Quantity One software (Bio-Rad, Mississauga, ON, Canada) and expressed as a ratio [ABCG2 vs. glyceraldehyde 3-phosphate dehydrogenase (GAPDH)].
Western blotting. The ABCG2 protein expression level was detected by western blotting. Each cell line was grown to confluence, trypsinized, transferred to eppendorf tubes and rinsed with ice-cold PBS. The contents of each tube were suspended in 200 µl lysis buffer in the presence of protease inhibitors. The cell suspension was incubated for 20 min at 4˚C and centrifuged for 10 min at 32,000 x g to obtain a clear supernatant. The protein concentration was measured by the Bio-Rad protein assay with bovine serum albumin (BSA) as a standard (Bio-Rad Laboratories, Mississauga, ON, Canada). For the western blot analysis, the protein extract was analyzed using SDS gel electrophoresis on appropriate polyacrylamide gels (5%) with 50 µg protein loaded on each lane. The proteins on the gels were then transferred to polyvinylidene difluoride (PVDF) membranes by a semi-dry transfer method. Subsequent to blocking with Tris-buffered saline (TBS) containing 5% skimmed milk and 0.1% Tween-20, the membranes were probed with a primary antibody against ABCG2 (mouse monoclonal antibody, clone no. sc-18841; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The antibody was used at a dilution of 1:1,000. The membrane blots were then reacted with a secondary antibody (horseradish peroxidase anti-mouse immunoglobulin G), washed extensively with TBS (0.1% Tween-20) and submerged in 3,3'-diaminobenzidine (DAB), then developed to visualize the antibody-antigen complexes.
Statistical analysis. The statistical analysis was performed using SPSS 11.5 software (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± SD and three individual experiments were performed in triplicate. A one-way ANOVA and an LSD test were used to compare the data between three or more groups. P<0.05 was considered to indicate a statistically significant difference.

Results
Establishment of Eca109/ABCG2 cell line. The Eca109 cells that were transfected with PCDNA3.1-ABCG2 plasmids were selected by G418 for 14 days. The positive clones, i.e. the Eca109/ABCG2 cells, survived, but the negative clones died (Fig. 1). The Eca109 cells that were transfected with PCDNA3.1, i.e. the Eca109/PCDNA3.1 cells, were used as the control cells.
The expression of ABCG2 in the Eca109, Eca109/ABCG2 and Eca109/PCDNA3.1 cells was examined using RT-PCR, western blot analysis and flow cytometry. The ABCG2 expression level in the Eca109/ABCG2 cells was higher than that in the Eca109 and Eca109/PCDNA3.1 cells (P<0.05; Fig. 2; Table I).
Resistance of Eca109/ABCG2 cells to anticancer agents. Following the anticancer drug treatment using ADM, DNR   (Table II).
ADM efflux effect of Eca109/ABCG2 cells assayed by flow cytometry. To investigate how the Eca109/ABCG2 cells resisted the anticancer agents, the ADM efflux effect was investigated using flow cytometry (Fig. 3). The cells were incubated at 37˚C with 0.02 µg/ml ADM for 2 h and then without ADM for 1 h.
The level of ADM decreased more in the Eca109/ABCG2 cells than in the Eca109 and Eca109/PCDNA3.1 cells. The ADM efflux effect of the Eca109/ABCG2 cells was more than that of the Eca109 and Eca109/PCDNA3.1 cells.

Reversal of drug resistance by Art in Eca109/ABCG2 cells.
The ability of Art to reverse the drug resistance of the Eca109/ABCG2 cells was examined using an MTT assay (Table III). Art alone at 0.01, 0.1 and 1 µmol/l had no cytotoxic effects on the Eca109, Eca109/PCDNA3.1 and Eca109/ABCG2 cells. Therefore, 0.01, 0.1 and 1 µmol/l Art was used in this experiment. Art at

Effect of Art on the apoptosis of Eca109/ABCG2 cells induced by ADM.
To investigate the reversal of the drug resistance caused by Art in the Eca109/ABCG2 cells, the effect of Art on the apoptosis of the Eca109/ABCG2 cells induced by ADM was investigated. The apoptosis rate of the Eca109/ABCG2 cells following the treatment with ADM and Art was higher than that following the treatment with ADM alone (P<0.05; Fig. 4; Table IV).

Effect of Art on the cellular accumulation and efflux of ADM.
To investigate how Art reversed the resistance of the Eca109/ABCG2 cells to the anticancer agents, the effect on the accumulation of ADM was investigated using flow cytometry. The increased accumulation of ADM in the Eca109/ABCG2 cells caused by Art was due to the inhibition of the ADM efflux (Fig. 5). When the cells were incubated at 37˚C with 0.02 µg/ml ADM for 2 h and then without ADM and with or without 1 µmol/l Art for 1 h, the level of ADM in the Eca109/ABCG2 cells with Art showed a smaller decrease than that without Art. Art inhibited the efflux of ADM from the Eca109/ABCG2 cells.

Effect of Art on the expression level of ABCG2 in Eca109/ABCG2 cells.
To investigate the mechanism of the reversal of resistance caused by Art in the Eca109/ABCG2 cells the effect of Art on the expression level of ABCG2 in the Eca109/ABCG2 cells was investigated. Art inhibited the expression level of ABCG2 protein in the Eca109/ABCG2 cells (Fig. 6).

Discussion
Chemotherapy is the most common treatment for esophageal carcinoma, particularly for advanced esophageal cancer and       recurrent cancer. However, the response from patients with recurrent esophageal cancer remains poor. Drug resistance is major obstacle for chemotherapy results and is attributable to several processes that occur in neoplastic cells. One of these processes is the decreased accumulation of anticancer drugs within the cancer cells due to drug efflux, which is mediated by ABC transporters (19,20). Overexpression of these transporters results in drug resistance in a number of tumors (21,22). However, the significance of the expression of the ABC protein in esophageal cancer has not yet been reported. The present study investigated a potential correlation between ABCG2 expression and MDR in esophageal cancer. ABCG2 is a member of the ABC family and functions as a ABC discharge pump. ABCG2 was initially isolated from a doxorubicin-resistant MCF-7/AdrVP breast cancer cell line (21).
In order to investigate the correlation between drug resistance in esophageal cancer and ABCG2 expression, the ABCG2 gene was transfected into the Eca109 esophageal cancer cell line to establish Eca109/ABCG2 cells. The expression level of ABCG2 in the Eca109/ABCG2 cells was higher than that in the Eca109 and Eca109/PCDNA3.1 cells (Fig. 2). The Eca109/ABCG2 cells showed cross-resistance to ADM, DNR and MIT compared with the Eca109 and Eca109/PCDNA3.1 cells (Table I). The drug efflux effect of the Eca109/ABCG2 cells was stronger than  that of the Eca109 and Eca109/PCDNA3.1 cells (Fig. 3). The Eca109/ABCG2 cell line was multidrug resistant. A high expression of ABCG2 in the cells enhanced the drug efflux effect of ADM, DNR and MIT. Therefore increasing the expression level of ABCG2 resulted in MDR. Resistance reversal agents for the ABC protein have been studied and due to the greater toxicity, few agents are used in clinical applications. The identification of a reversal agent with low toxicity and efficient resistance is required. Art is a derivative of artemisinin. Studies have identified that artemisinin and its derivatives are anti-inflammatory, antifibrosis, fight schistosomiasis and antitumor agents. Art is a good antimalarial drug, particularly for heavy and drug-resistant malaria, however, studies have paid more attention to the antitumor function of Art (13)(14)(15)(16). The antitumor effect of Art has not produced cross resistance (17), indicating that Art confers a reversal to drug resistance. The present study investigated the function and mechanism of Art in the reversal of esophageal cancer drug resistance. The esophageal cancer drug resistant Eca109/ABCG2 cell line was established by transfecting the ABCG2 gene into Eca109 cells. The Eca109/ABCG2 cells had a high expression of the ABCG2 gene and protein compared with the Eca109 cells (Fig. 2). High ABCG2 expression in the cells resulted in MDR. The Eca109/ABCG2 cells were resistant to ADM, DNR and MIT (Table I). The mechanism of MDR that was induced by ABCG2 was detected for further investigation. The ADM content in the Eca109/ABCG2, Eca109/PCDNA3.1 and Eca109 cells was identified using flow cytometry following the treatment with ADM. The ADM content in the Eca109/ABCG2 cells was lower than in the Eca109/PCDNA3.1 and Eca109 cells (Fig. 3), indicating that the drug efflux effect of Eca109/ABCG2 was stronger than that of the other two types of cells. The Eca109/ABCG2 cells were a cell line with an ABCG2 drug-resistant phenotype. ABCG2-mediated drug secretion reduced the level of anticancer drugs in the Eca109/ABCG2 cells, which caused MDR.
The inhibitory effect of ADM on the Eca109/ABCG2 cells was enhanced by combining the drug with Art (Table II). The apoptosis rate of the Eca109/ABCG2 cells that was induced by ADM was also enhanced by combining the drug with Art (Fig. 4). These results suggested that Art had a role in the reversal of drug resistance. For the next level of investigation, the mechanism behind the reversal of drug resistance by Art was investigated. Art reduced the level of ABCG2 expression (Fig. 6) and inhibited the drug efflux effect (Fig. 5) of the Eca109/ABCG2 cells. Art was able to reverse drug resistance by reducing ABCG2 expression and increasing the anticancer drug concentration in the cancer cells.
In summary, ABCG2 expression participated in the MDR of esophageal cancer. Art was able to reverse the drug resistance by reducing ABCG2 expression and increasing the anticancer drug concentration in the cancer cells. Art is expected to be associated with low toxic side-effects and may be a highly efficient resistance reversal agent in the clinic.


extfieldsofstudy:
Biology
Medicine
Computer Science

text:
Using Protein Clusters from Whole Proteomes to Construct and Augment a Dendrogram

In this paper we present a new ab initio approach for constructing an unrooted dendrogram using protein clusters, an approach that has the potential for estimating relationships among several thousands of species based on their putative proteomes. We employ an open-source software program called pClust that was developed for use in metagenomic studies. Sequence alignment is performed by pClust using the Smith-Waterman algorithm, which is known to give optimal alignment and, hence, greater accuracy than BLAST-based methods. Protein clusters generated by pClust are used to create protein profiles for each species in the dendrogram, these profiles forming a correlation filter library for use with a new taxon. To augment the dendrogram with a new taxon, a protein profile for the taxon is created using BLASTp, and this new taxon is placed into a position within the dendrogram corresponding to the highest correlation with profiles in the correlation filter library. This work was initiated because of our interest in plasmids, and each step is illustrated using proteomes from Gram-negative bacterial plasmids. Proteomes for 527 plasmids were used to generate the dendrogram, and to demonstrate the utility of the insertion algorithm twelve recently sequenced pAKD plasmids were used to augment the dendrogram.

Introduction
The availability of complete proteomes for hundreds of thousands of species provides an unprecedented opportunity to study genetic relationships among a large number of species. However, the necessary software tools for handling massive amounts of data must first be developed before we can exploit the availability of these proteomes. Currently the tools used for clustering either are restricted in terms of the number of proteomes that can be examined because of the time required to obtain results or else are restricted in terms of their sensitivity. For example, clustering by means of hidden markov models (HMM), multiple sequence alignment, and pairwise sequence alignment by means of the Smith-Waterman alignment algorithm are limited by their time complexity. The Smith-Waterman algorithm, a dynamic programming algorithm, is known to give optimal alignment between two protein sequences for a given similarity matrix [1], but alignment of two sequences of lengths and requires ( ) time. On the other hand, heuristic approximate alignment methods, frequently based on BLAST and its variants [2], reduce the computational time required; for example, in practice BLAST effectively reduces the time to ( ), but this comes at the risk of losing sensitivity to homology detection. In fact, numerous articles-for example, see [3,4]-have discussed this loss of sensitivity in BLAST-based results compared to those of the Smith-Waterman algorithm. To ensure that a maximum number of homologous sequences are identified, highly sensitive pairwise homology detection is required. Otherwise, the clusters of homologous sequences Protein profiles obtained by means of a given clustering method will not include all possible members and, ultimately, the final results will be less accurate.
In this work we use an alternative sequence comparison algorithm and clustering method called pClust. Rather than approximating Smith-Waterman, pClust systematically eliminates sequence pairs with little likelihood of having alignments and then only employs the Smith-Waterman algorithm on promising pairs [5]. Clustering is accomplished using a method based on a previously developed approach called shingling [6]. By filtering out unlikely sequences and using the Smith-Waterman algorithm judiciously, pClust remains highly sensitive to sequence homology without loss of speed. In an unpublished study of 6,602 proteins from four bacterial proteomes, pClust and BLAST results were compared, and BLASTp missed more than 69% of the aligned pairs identified by pClust. In a different study, a direct clusters-to-clusters comparison was performed with BLAST results used as the test and pClust results used as the benchmark [7]. The results showed that all the BLAST results were included within the pClust results but BLAST missed 14% of the clustered pairs obtained with pClust. In addition to its sensitivity and speed, pClust is readily parallelizable, and to cluster proteins from the proteomes of thousands of species will require highperformance computing platforms and the use of parallel algorithms.
This work was initiated by our interest in plasmids. We wanted a software tool that would allow us to obtain genetic relationships among 527 Gram-negative bacterial plasmids based on their putative proteome sequences. In addition, we wanted an efficient means of adding new plasmids to our initial dendrogram as their proteomes become available. Plasmids are typically circular DNA sequences that can transfer between and replicate within bacteria and that are generally classified as broad-or narrow-host range [8,9]. Plasmid sequences are described as mosaic because they are composed of DNA arising from many sources [10]. Plasmids serve to shuttle important adaptive traits, such as antibiotic resistance, between organisms [11,12]. Consequently, understanding the genetic relationships among plasmids is important, for example, in the study of microbial evolution, in medical epidemiology, and in assessing the dissemination of antibiotic resistance genes [13,14]. There are a number of approaches to examine plasmid relationships. Some researchers focus on the identification of important plasmid backbone genes that are involved in horizontal gene transfer (HGT) or replication within bacterial hosts [15,16]. Some approaches compare compositional features such as genomic signatures and codon usage [5,17]. Some researchers use network-based representations to explore genetic relationships among plasmids [5,18,19]. In this work we use the whole proteomes of 527 Gramnegative (GN) bacterial plasmids to construct a dendrogram.
We use protein cluster information from pClust to construct our dendrogram and then to predict the relationship of new plasmids within the structure of this tree. A binary profile is created for each species, indicating the presence or absence of a protein in each cluster ( Figure 1). The concatenation of all the profiles results in a binary matrix from which a distance matrix is calculated, and neighbor joining is then used to construct a dendrogram. The binary matrix also can be viewed as a library of individual profiles that can serve as correlation filters for a new taxon. A profile for a new taxon can be quickly correlated with the profiles in the library to filter out the profile with the highest correlation coefficient. This correlation coefficient is then evaluated based on known biological information and a decision is made as to whether Correlation filter library should be classified near the largest coefficient plasmids ⋅ Insert into original tree the taxon should be added to the tree. If it is to be added, its binary profile is added to the binary matrix, a new distance matrix is calculated, and neighbor joining is again used to construct a new dendrogram with the additional taxon. To utilize the algorithm for new plasmids, we focus on sequences from twelve pAKD plasmids that were isolated from Norwegian soil [20]. These plasmids belong to incompatibility groups IncP-1( ) and IncP-1( ). A phylogenetic tree constructed using multiple alignment of the relaxase gene traI is presented by Sen et al. [20] and serves as a basis of comparison for our augmentation results.

Data Preparation.
Zhou et al. [21] presented a virtual hybridization method to construct a dendrogram for 527 GN bacterial plasmids with 50 or more putative coding genes. The same plasmids are used in this study to facilitate comparison. BLASTp with default parameters was used to remove duplicate proteins within plasmid sequences using a similarity score defined by the formula (length of matching sequence) * (BLAST identity score)/(length of reference protein + length of matching sequence) ≥0.45-that is, proteins with scores ≥0.45 were considered to be duplicates [22]. The maximum score 0.5 is obtained when two proteins are an exact match. Including the matching sequence length in the denominator of the formula insures that a large difference in sequence lengths does not bias the results. After removal of duplicate proteins, more than 97,000 protein sequences remained.

Dendrogram Construction.
The flowchart in Figure 1 shows the approach used to construct a dendrogram for the plasmids based on the >97,000 plasmid protein sequences. The protein sequences 1, 2, . . . , are used as input into the pClust program [5], which employs the Smith-Waterman algorithm to perform pairwise comparison of a subset of the sequences. The output from pClust is composed of clusters 1, 2, . . . , of homologous proteins. Protein profiles 1, 2, . . . , are then created for all the plasmids from the pClust output files. Each profile consists of a binary sequence with 1 indicating the presence of a protein and 0 indicating absence (Figure 1). The pClust software was used with default settings in the configuration file except for Exact-MatchLen for which a value of 4 was used. A total of 6,618 clusters (defined as having at least two proteins) were identified by pClust. The resulting 527 × 6, 618 binary matrix was used to construct the dendrogram for two different distance measures. The Jaccard distance metric was originally developed for computation with binary matrices and is given by where is the number of clusters 1, 2, . . . , that are 1 for species and 0 for species , is the number of clusters that are 0 for species and 1 for species , and is the number of  clusters that are 1 for both species and . We also employ a conventional Euclidean distance metric. For both metrics, a neighbor-joining algorithm was used to obtain the final dendrogram.  plasmids into the existing tree structure as described in this section, where execution of the insertion algorithm takes only a few minutes on a laptop computer.

Insertion of New
To insert a new plasmid into an existing dendrogram, proteins 1, 2, . . . , from a new plasmid are extracted from the plasmid proteome ( Figure 2). BLASTp is performed with these proteins against all the proteins in the 6,618 clusters to determine the protein profile for the new plasmid. A protein is considered to be a member of a cluster when its similarity score is >0.2. The similarity score is given by (length of matching sequence) * (BLAST identity score)/(length of reference protein + length of matching sequence). The cutoff value of 0.2 is consistent with the 40% sequence similarity used as a parameter setting in pClust. Correlation filtering is then performed with the correlation filter library consisting of the protein profiles of the original 527 GN bacterial plasmids. The Pearson's product-moment correlation coefficient, whose absolute value is less than or equal to 1, is used to measure the correlation between two profiles [23,24]. The larger the correlation value, the greater the similarity between  two profiles. This value is used to determine whether the plasmid fits into the dendrogram and, if so, where it should be located as explained in the discussion section. When appropriate, the new protein profile is added to the binary matrix, and a tree is constructed from the entire matrix as described in the previous section.

Results and Discussions
3.1. 527-Plasmid Dendrogram. Following the procedure described above, a dendrogram was constructed for 527 GN bacterial plasmids. Because of its size, it is not shown, but it is available as supplementary information in Newick standard format (.nwk) for both Jaccard and Euclidean distance metrics and can be viewed using MEGA5 [25]. A tree constructed using the Jaccard distance metric for the same subset of 50 plasmids used in [21] is shown in Figure 3, and the Euclidean distance version is shown in Figure 4. These trees are very similar with only a slight difference in the clustering of the Borrelia plasmids. The tree constructed using the Euclidean distance metric is closer to the one shown in [21], but the Jaccard tree does a better job of clustering the Borrelia plasmids [26,27]. The Jaccard distance metric is commonly used for a binary matrix. Nevertheless, the results based on Euclidean distance compare favorably with those obtained for a nonbinary intensity matrix using a different approach [21]. It is not clear which distance method gives more accurate results so users should use both matrices and the decision as to which one is more accurate should be determined on the basis of the biology of the system.

Insertion of New Plasmids.
We applied our correlation filter algorithm to twelve new plasmids from the pAKD family [20]. The twelve plasmids cluster together and are most closely grouped with genera typical of other soil bacteria. The correlation coefficient values among the pAKD plasmids were >0.7 and decreased relative to the other plasmids with distance to >0.5 ( Figure 5). pAKD plasmids 16, 25, and 34 belong to the IncP-1( ) compatibility group and form a discrete cluster: pAKD plasmids 1, 14,15,17,18,29,31, and 33 cluster as the IncP-1( ) compatibility group. Although pAKD26 falls into the IncP-1( ) clade, it should be in the IncP-1( ) group if compatibility grouping is considered the gold standard for comparison. Nevertheless, the placement is distal from the eight other plasmids in the group, and pAKD26 was actually designated as IncP-1 -2 to differentiate it from the other eight plasmids as recently described in [28]. Our results are consistent with [20].
Importantly, the correlation coefficient is used to check the final dendrogram-that is, a new plasmid should be located near the plasmid with which it is most highly correlated. In addition, the correlation coefficient is used to determine whether a plasmid should even be inserted into a dendrogram. In other words, how does the magnitude of the correlation coefficient influence our confidence in the placement of a new plasmid within an existing dendrogram? Several works offer guidelines for the interpretation of a correlation coefficient [29,30], but all criteria are in some way arbitrary and ultimately interpretation of a correlation coefficient depends on the purpose. In our case, we chose a value of 0.5, but we also require biological evidence-for example, that a plasmid is, in fact, from a GN bacterium.
To further examine the correlation coefficient, we randomly selected 10 Gram-positive bacterial plasmid proteomes from 10 different genera. The correlation coefficients were found to range from 0.112 to 0.234. GP bacterial plasmids do not belong in our GN bacterial plasmid dendrogram, and our minimum correlation value of 0.5 suffices to exclude these unrelated plasmids. While this level of discrimination is easy to identify, we should note that the 527 GN bacterial plasmids considered in this study do not represent the full diversity of GN plasmids. Thus, it is possible to obtain a small correlation coefficient value for a completely new and uncharacterized GN plasmid. If the new plasmid is able to meet an underlying correlation threshold, it can be placed within the dendrogram structure, and by incorporating the new plasmid sequence information into the correlation filter library, we can group future plasmids that may be closely related to it.
While the method of inserting new plasmids into an existing tree is fast and efficient, at some point, generation of a new dendrogram using all proteins from all the taxa will probably be required. We do not know at what point this will occur, but we assume it will be necessary eventually to insure that all possible protein clusters are included. Recall that a cluster must contain at least two proteins to be considered a cluster. Thus, any new plasmid containing a protein that would have formed a cluster with a single discarded protein represents incomplete information in the library. It is probable that the total number of clusters for all Gram-negative plasmids will ultimately be much greater than 6,818.

Conclusion
In this work we present a new ab initio method for constructing a dendrogram from whole proteomes that begins with output from pClust, a software program developed for homology detection for large-scale protein sequence analyses. We develop an efficient approach for insertion of a new species into the dendrogram based on the use of a correlation filter library. This is much more efficient than constructing an entirely new tree which is computationally costly. We illustrate our method by creating a dendrogram for 527 Gram-negative bacterial plasmids and augmenting this dendrogram with twelve pAKD plasmids isolated from Norwegian soil. For purposes of comparison, we also construct a smaller dendrogram consisting of 50 species and use two different distance metrics. The two resulting trees agree well with results shown in [21]. The classification results for the twelve plasmids agree with a phylogenetic tree constructed using multiple sequence alignment of the relaxase gene traI presented in [20].


extfieldsofstudy: Biology Medicine

text:
Low ABCB1 Gene Expression Is an Early Event in Colorectal Carcinogenesis

The ABCB1/MDR1 gene product ABCB1/P-glycoprotein is implicated in the development of colorectal cancer (CRC). NFKB1 encodes transcription factors regulating expression of a number of genes including ABCB1. We have previously found association between the ABCB1 C-rs3789243-T polymorphism and CRC risk and interactions between the ABCB1 C-rs3789243-T and C3435T polymorphisms and meat intake in relation to CRC risk (Andersen, BMC Cancer, 2009, 9, 407). ABCB1 and NFKB1 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 101 adenoma cases (12 with severe dysplasia, 89 with mild-moderate dysplasia) and from 18 healthy individuals, together with gene polymorphisms in ABCB1 and NFKB1. ABCB1 mRNA levels were highest in the healthy individuals and significantly lower in mild/moderate and severe dysplasia tissue (P<0.05 for both), morphologically normal tissues close to the tumour (P<0.05), morphologically normal tissue at a distance from the tumour (P<0.05) and CRC tissue (P<0.001). Furthermore, ABCB1 mRNA levels were lower in adenomas and carcinomas compared to morphologically normal tissue from the same individuals (P<0.01). The ABCB1 C-rs3789243-T and NFKB1 -94ins/del homozygous variant genotypes were associated with low ABCB1 mRNA levels in morphologically normal sigmoid tissue from adenoma cases (P<0.05 for both). NFKB1 mRNA levels were lower in both tumour and normal tissue from cancer patients (P<0.001) as compared to healthy individuals but we were unable to show association between NFKB1 -94ins/del genotype and NFKB1 mRNA levels. This study suggests that low ABCB1 mRNA levels are an early event in CRC development and that the two polymorphisms affect ABCB1 mRNA levels whereas low NFKB1 mRNA levels occur later in carcinogenesis. Low ABCB1 protein levels may promote colorectal carcinogenesis through increasing intracellular exposure to carcinogenic ABCB1 substrates.

Introduction
Colorectal cancer (CRC) constitutes the second most common cancer in the Western world and the prevalence is expected to increase due to demographic trends and adaption to westernized lifestyle in developing countries. In the Western world, one in 20 will develop CRC before the age of 75. ABCB1/P-glycoprotein is a membrane protein, encoded by ABCB1/MDR1, which transports substrates from the enterocytes to the intestinal lumen, thereby, restricting the exposure of the enterocytes to the substrates of the ABCB1 transporter. Known substrates include various xenobiotics and endogenous compounds such as cholesterol, IL-1b, IL-2, IL-4, and IFNc [1]. The ABCB1 has been implicated in intestinal carcinogenesis in animal models and in epidemiological studies. Abcb1/mdr1a knock-out mice develop colitis and later intestinal adenocarcinomas [2] suggesting that the absence of ABCB1 confers risk of inflammation-related CRC. In human, ABCB1 polymorphisms are associated with risk of CRC in some but not all studies [3][4][5][6]. The ABCB1 intron 3 C-rs3789243-T is a marker polymorphism and was found to be associated with risk of CRC in a prospective, population-based study (OR = 1.52, 95%CI 1.12-2.06) [3]. Similarly, a metaanalysis of case-control studies found an increased risk of CRC among carriers of the combined C3435T and G2677T/A wildtype alleles in Caucasians (OR = 1.22, 95%CI 1.03-1.44). In a subsequent study, the C3435T polymorphism was assessed in three large cohorts [5]. The C3435T C-allele was associated with a low risk of CRC (OR = 0.79, 95%CI 0.66-0.96) in the cohort from Southwest Germany (1809 cases) and a high risk, although not statistically significant, in the cohort from Northern Germany (2169 cases) whereas no change of risk was found in the Czech cohort (699 cases) [5].
ABCB1 levels in solid tumours have been determined in a few studies [7][8][9]. ABCB1 protein levels were found to be low in CRC tissue compared to well-differentiated tissue in archival material from 51 cancer patients estimated by immunohistochemistry [8]. Also, ABCB1 mRNA and protein levels were found to be lowered in renal cell carcinoma tissue compared to normal looking cortex in 82 nephrectomised cancer patients [7]. In the German study, the variant alleles of C2677T/A, and G3435T polymorphisms were associated with high ABCB1 mRNA levels in normal renal tissue [7]. In contrast, the variant alleles of 2677T and 3435T polymorphisms were associated with low mRNA levels in 73 normal liver tissue sample from Chinese patients [10]. Although intestinal ABCB1 transport activity can be determined using a model substrate [11], this has not been done on cancer cells or cancerous tissue.
The MDR1 gene has a TATA box-less promoter regulated by a large number of factors including pregnane X receptor (PXR) and nuclear factor-kB (NF-kB). The NFKB1 -94ins/del polymorphism is a four nucleotide insertion/deletion polymorphism in the promoter region of NFKB1 leading to lower transcription levels and lower protein levels of the NF-kB subunits p50 and p105 [12]. Genetically determined low NF-kB p50 levels were associated with an increased risk of CRC among Danes, whereas genetic variation in PXR was not associated with CRC risk [13].
In the present study our aim was to assess the intestinal levels of ABCB1 and NFKB1 mRNA in adenoma and CRC cases and in control subjects in order to characterize the role of ABCB1 in the development of CRC. Furthermore, we assessed the impact of genetic variants in ABCB1 and NFKB1 on the ABCB1 mRNA levels in morphologically normal and affected colon tissues.

Study Cohort
The KAM (Kolorektalkreft, arv og miljø) cohort is based on the screening group of the Norwegian Colorectal Cancer Prevention study (the NORCCAP study) in the county of Telemark and a series of clinical CRC cases operated at Telemark Hospital (Skien) and Ulleval University Hospital (Oslo) [14,15]. In short, 20,780 healthy men and women, 50-64 years of age, drawn at random from the population registry in Oslo (urban) and the county of Telemark (mixed urban and rural) were invited to have a flexible sigmoidoscopy screening examination. The KAM cohort is based on an ethnically homogeneous group of Norwegian origin.
The KAM biobank consists of samples from individuals with adenomas in the large intestine (991 adenomas and 53 hyperplastic polyps), 234 cases with CRC and 400 controls, defined as individuals with normal findings at flexible sigmoidoscopy screening. The study was performed in accordance with the Helsinki Declaration. The Regional Ethics Committee and the Data Inspectorate approved the KAM study (S-98190, 2009/ 2021). The ID number for the study is NCT00119912 at ClinicalTrials.gov. All participants gave verbal and written informed consent.

Biological Material
In the present study, blood samples were available from 167 cases with carcinomas, 990 cases with adenomas and 400 controls. Intestinal tissue was available from 121 cases with carcinoma, 100 cases with adenomas and 18 controls with normal endoskopic findings. From individuals with adenomas, control tissue was sampled 30 cm above the anus. From patients with carcinomas, two samples of morphologically normal tissue were taken from the surgical specimen. One sample was taken adjacent to the cancer (normal tissue 2) and the other sample was taken as distant from the cancer as possible (normal tissue 1). Complete samples were available from 75 cases with mild-moderate dysplasia, 11 cases with severe dysplasia, and 99 CRC cases. The histology of the adenomas was examined independently by two histopathologists, who categorised the degree of dysplasia as either mild/moderate (n = 87) or severe (n = 13). Consensus was reached in all cases. Carcinomas were classified according to Dukes staging.  Primers were designed within different exons and with the probe covering an exon-exon border to prevent amplification of genomic DNA (ABCB1) or with a primer covering an exon-exon border (NFKB1). Primers and probes were obtained from TAGCopenhagen (Denmark). The endogenous b-actin control was obtained using a pre-developed assay (part.no.4310881E) from Applied Biosystems. In a validation experiment, a dilution series was assayed by the comparative C t method [16]. The assays were quantitative over a range of 128-fold dilution. Samples were quantified in triplicates. The standard deviation of triplicates was 6% or less. The standard deviation on repeated measurements of the same sample (the control) in separate experiments was 16% (ABCB1) and 13% (NFKB1), indicating the day-to-day variation of the assay. Negative controls (where the RNA was not converted into cDNA) and positive controls were included in all runs. Samples for which either the b-actin, ABCB1, or NFKB1 values fell outside the upper or lower limits of the standard curve were excluded from the study.

Genotyping
Genomic DNA was isolated from blood samples according to standard procedures. ABCB1 C-rs3789243-T genotyping data was retrieved from Andersen et. al. [6]. ABCB1 C3435T (rs1045642) and NFKB1 -94ins/del (rs28362491), were genotyped by KBioscience (kbioscience.co.uk). For ABCB1, C was considered as the wildtype allele even though it was not the major allele, since this is the commonly used nomenclature.

Statistics
GraphPad Prism 4 was used for the statistic calculations. The data were not adjusted for gender since the incidence ratio of CRC between the genders is 1:1 in Norway. Kruskal Wallis and Dunns Multiple Comparison test was used to compare the mRNA level of all intestinal tissues and Paired Student's t-test was used for comparison of affected tissue and morphological normal tissue from the same individual.

ABCB1 mRNA Levels in Intestinal Tissue
Study participants are described in Table 1. ABCB1 mRNA levels were significantly lower in dysplastic tissue (P,0.01), morphologically normal tissues 1 and 2 from cancer patients (P,0.01, for both) and tumour tissue (P,0.001) compared to healthy individuals (Figure 1). In order to elucidate whether ABCB1 mRNA levels are diminished as an early event in colorectal carcinogenesis, individuals with adenomas were further subdivided into mild/moderate dysplasia (N = 89) and severe dysplasia (N = 12) ( Table 2). Table 2 shows a low level of ABCB1 mRNA already in mild/moderate dysplasia which is maintained at a low level throughout carcinogenesis. Comparing ABCB1 mRNA levels, a significantly lower mRNA level was found in the mild/moderate adenoma, severe adenoma and CRC tissues ( Table 2) as compared to morphologically normal tissue from the same individuals. There was no correlation between the ABCB1 mRNA levels and age, gender or Duke's stage of the carcinoma (Dukes stage A (n = 19), stage B (n = 47), and stage C (n = 29)). Figure 2 shows ABCB1 mRNA levels in intestinal adenoma cases (left panel) and carcinoma cases (right panel) subdivided by ABCB1 C-rs3789243-T, C3435T, and NFKB1 -94ins/del genotypes, respectively. In morphologically normal sigmoid tissue from the intestinal adenoma cases, significantly lower ABCB1 mRNA levels were found for carriers of the ABCB1 C-rs3789243-T variant homozygous genotype compared to the homozygous wildtype carriers (P,0.05). Furthermore, the NFKB1 del/del genotype was associated with low ABCB1 mRNA level compared with the heterozygous genotype (P,0.05). Similar trends, however not  statistically significant, were found in adenoma tissue. Among carcinoma cases, no associations between any genotypes and ABCB1 mRNA levels were found in any tissue (Figure 2, right panel). These data suggest that NFKB1 play a role in regulation of the expression of ABCB1 gene in normal tissue as represented by the morphological normal sample from individuals with dysplasia but not in cancerous tissue or normal tissue from cancer patients. The NFKB1 mRNA levels were therefore determined in all intestinal samples in order to further investigate this.

NFKB1 mRNA Levels in Intestinal Tissue
Generally the NFKB1 mRNA levels were significantly lower in tissues from cancer patients but unaffected in individuals with dysplasia. NFKB1 mRNA levels were significantly lower in morphologically normal tissues 1 and 2 from cancer patients, and tumour tissue (P,0.001 for all) compared to healthy individuals ( Figure 3). We found no difference between affected and non-affected tissue in individuals with dysplasia or cancer patients (Table 3). There was no correlation between the NFKB1 mRNA levels and age, gender, or Duke's stage of the carcinoma. subdivided by the NFKB1 -94ins/del genotype. We found no significant difference in the NFKB1 mRNA level depending on the NFKB1 -94ins/del genotype.

Discussion
In the present study, we found that intestinal tissue from healthy individuals had the highest ABCB1 mRNA level. Apparently morphologically normal tissue from cancer cases had significantly but only slightly lowered ABCB1 mRNA levels. In contrast, the mRNA levels in both adenoma tissue and carcinoma tissue were decreased by half compared to the mRNA level found in the healthy controls. ABCB1 mRNA levels were also lower in adenomas and carcinomas compared to morphologically normal tissue from the same individuals (Table 2).
Our findings on carcinomas are in agreement with previous findings by Judicibus et al. on CRC and Haenisch et al. on renal cell carcinomas, who both found low ABCB1 mRNA and/or protein levels in tumour tissue compared to non-affected tissue surrounding the tumour (10;11). We extend the study by Judicibus et al., by analysing tissue from healthy individuals and individuals with adenomas.
The presence of a reduced ABCB1 mRNA level already in mild/ moderate dysplastic tissue (Table 2) suggests that lowered ABCB1 mRNA level is an early event in colorectal carcinogenesis. Therefore, assuming that a lowered level of ABCB1 mRNA results in low ABCB1 activity in the adenomas, this would lead to a higher intracellular exposure to various carcinogenic substrates of the ABCB1 transporter. The finding that once CRC has developed, low ABCB1 mRNA levels were found in both carcinoma tissue and morphologically normal tissue surrounding the tumour, could suggest that low ABCB1 mRNA precede tumour formation or that the presence of tumour tissue affects gene expression in the surrounding tissue.
We evaluated the association between genetic variants in ABCB1 and NFKB1, and intestinal ABCB1 mRNA levels. ABCB1 C-rs3789243-T and NFKB1 -94ins/del homozygous variant genotypes were associated with lowered ABCB1 mRNA levels in morphologically normal tissue from adenoma cases. The ABCB1 gene is highly polymorphic and extensive linkage between  polymorphisms make identification of causal variants difficult [17]. Thus, it is most likely that the polymorphism is biologically nonfunctional and the detected association with ABCB1 mRNA levels is caused by linkage to one or more functional polymorphisms [17]. The studied NFKB1 ATTG ins/del polymorphism is a functional deletion in the promoter region of NFKB1 encoding the p50 and p105 subunits of NF-kB. The deletion abolishes a transcription factor binding site in the promoter region and leads to lower NF-kB p50 synthesis [12]. However, we found no association between NFKB1 ins/del genotype and NFKB1 mRNA levels. It seems contradictory that we are able to find association between the NFKB1 -94ins/del polymorphism and ABCB1 mRNA levels but not with NFKB1 mRNA levels. The reason might be that even relatively small differences in p50 protein levels may be sufficient to change transcription rates of target genes as shown by Kaban et al. [12]. Alternatively, it may be due to the relative low number of individuals analysed. Transcription of C-reactive protein is known to be activated by the NF-kB p50 dimer and the NFKB1 ins/del polymorphism was previously shown to be associated with blood levels of C-reactive protein in a study group of more than 1800 women [18]. Furthermore, our results suggests that the observed low ABCB1 mRNA level in adenoma tissue must be caused by changes is the concentration of transcription factors other than NF-kB p50 as NFKB1 mRNA levels was unchanged in adenoma tissue.
In individuals with adenomas, we found that carriers homozygous for the ABCB1 C-rs3789243-T and NFKB-94 deletion variant alleles have significantly lower ABCB1 mRNA levels in morphologically normal tissue suggesting that these polymorphisms cause lower ABCB1 mRNA levels. In contrast, ABCB1 mRNA levels in the carcinogenic tissue and the morphologically normal tissue surrounding the tumour are lowered independently of the studied polymorphisms in ABCB1 and NFKB1 (Figure 1 and 2) suggesting any regulation by these polymorphisms is lost once adenomas have been formed.
In a prospective Danish study, carriers of the NFKB1 -94 deletion and the ABCB1 intron 3 C-rs3789243-T variant alleles were at 1.45 and 1.55-fold higher risk of CRC, respectively, than carriers of the corresponding homozygous wildtype (95%CI 1.10-1.92, P = 0.03 and 95%CI 1.12-2.06, P = 0.03, respectively) [3,13]. In the same studies, interactions between ABCB1 and NFKB1 polymorphisms and intake of red and processed meat in relation to CRC risk were found [3,13]. ABCB1 C3435T homozygous C-allele carriers, ABCB1 C-rs3789243-T T-allele carriers and NFKB1 -94 deletion carriers were at high risk of CRC by meat intake (P for interaction = 0.02, 0.01, and 0.03, respectively).
At present, it is not clear how meat intake interacts with ABCB1 in relation to CRC development [19]. There are no known dietary carcinogens identified as ABCB1 substrates. Studies of abcb1 knock-out mice strongly suggest that abcb1 deficiency drives an inflammation-related carcinogenesis. The same mechanism may be important in human which is suggested by the identification of ABCB1 as a 'risk gene' in inflammatory bowel diseases [20].
This study used a case-control design. Because our hypothesis was biologically based, we did not correct for multiple analyses [21].A main strength of the study is the relatively large sample size. However, in the light of the obtained P-values and the number of statistical tests performed, we cannot exclude that some of our positive findings may be due to chance.
In conclusion, this study suggests that low ABCB1 mRNA expression is an early event in CRC development. ABCB1 mRNA levels are associated with genetic variation in ABCB1. Furthermore, low levels of NFKB1 encoding NFkB p50, which is involved in regulation of ABCB1 expression is a late event in carcinogenesis. Low ABCB1 levels may promote CRC by increasing intracellular exposure to carcinogenic or inflammatory ABCB1 substrates. Further studies on the functional role of ABCB1 in carcinogenesis are warranted.


extfieldsofstudy: Medicine Biology

text:
The RNA Binding Protein ESRP1 Fine-Tunes the Expression of Pluripotency-Related Factors in Mouse Embryonic Stem Cells

In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.

Introduction
Pluripotency is a unique state in which cells can self-renew indefinitely whilst maintaining the ability to differentiate into multiple cell types of the body. In embryonic stem (ES) cells, gene regulatory networks comprising of the core transcription factors, Oct4, Nanog and Sox2 as well as chromatin regulatory proteins are involved in pluripotency maintenance [1]. High endogenous levels of these factors are beneficial for ES cell pluripotency, but deregulated expression of pluripotency-associated transcription factors has been shown to change cell fate. Small increases or decreases in Oct4 expression promote the differentiation of ES cells into extraembryonic endoderm and mesoderm or trophectoderm, respectively [2,3]. Likewise, small increases in Sox2 can trigger the differentiation of ES cells into cells that express markers associated with a wide range of differentiated cell types [4]. Notably, there is increasing evidence for stemness and embryonic pathways reactivating during oncogenesis [5]. It is thus extremely important to deeper investigate the molecular mechanisms regulating the expression levels of the pluripotency factors.
The mechanisms that control the transcription of core pluripotency factors have been extensively studied [1,3,6,7,8]. In addition, several lines of evidence have recently underscored the importance of post-transcriptional regulation of gene expression in pluripotency maintenance [9]. To this regard, the action of microRNAs (miRNAs) as well as RNA-binding proteins (RBPs) involved in miRNA maturation, like dicer and dcgr8 has been investigated in detail [10,11,12,13]. More recently, the RBP Unr (Upstream of N-ras) has been reported to post-transcriptionally repress Gata6 expression, causing stabilization of the pluripotent state of ES cells [14]. L1TD1, which interacts with Lin28, is also required for human ES cells self-renewal [15]. Despite this progress, the full contribution of RNA and of RBPs (RNA-based) to the complex regulatory circuitry of pluripotency is probably still underestimated, as suggested by the recent discovery that the expression of many large intergenic non-coding (lincRNAs) has effect on ES cell gene expression and on their differentiation state [16,17]. In particular, the direct role of RBPs on the posttranscriptional regulation of core pluripotency factors expression in ES cells needs to be investigated in more depth. Genome-wide screenings aided by computational predictions may largely assist in this process [18].
To find new regulators of stemness/pluripotency, we analyzed conserved co-expression network (CCN) obtained from human and mouse stem-cell specific cDNA microarray data [19]. This analysis indicated the RBP Epithelial splicing regulatory protein 1 (Esrp1, also known as Rbm35a), first described as a tumor suppressor gene mutated in approximately 50% of primary colon tumors with microsatellite instability, as a protein possibly involved in pluripotency [20]. We found that depletion of ESRP1 in mouse ES cells resulted in increased self-renewal and impaired early differentiation in vitro. Moreover, ESRP1 binds to the mRNA of several pluripotency-related genes and decrease their polysomal loading, hence contributing to finely tune their expression levels in mouse ES cells. Altogether, our results indicate that ESRP1 is a new regulator of pluripotency.

Materials and Methods
Cell Culture and Differentiation E14 ES cells were cultured and differentiated in vitro in embryoid bodies (EBs) as previously described [21], [22]. Briefly, 300 ES cells were cultured in EB differentiation media (see File S1 for details) in ultra-low attachment 96-well plates (Corning). Two days later, the EBs were collected and further cultured in ultra low attachment 6-cm dishes for the indicated times. Mouse spermatogonial stem cells (SSCs) were isolated from juvenile mice testis and cultured as previously described [23]. Epcam-positive SSCs were prepared from adult mice testis by MACS sorting [24] and cultured on inactivated Mefs as previously described [25]. See File S1 for details on SSC cultures.

Generation of ES Cells with Stable Knockdown of ESRP1
Screening of short hairpin (Sh) RNA for efficient knockdown of Esrp1 in ES cells, vectors as well as lentiviruses production, quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting and immunofluorescence staining are described in File S1. Primers and probes employed for PCR and qRT-PCR are described in Table S1. Pluripotency, colony forming (alkaline phosphatase and methylene blue staining) and cell proliferation Figure 2. Pluripotency-related genes expression is affected by ESRP1 depletion. A. Confocal microscopy analysis of ESRP1 (red) in mouse ES cells reveal that the protein is expressed not only in the nucleus by also in the cytoplasm of these cells. Nuclei are stained with DAPI (blue). Scale bar is 5 mm. B. Representative Western blot analysis shows that ESRP1 is expressed mainly in the cytoplasm of mouse ES cells. As expected, Oct4 has a main nuclear localisation. Actin and Lamin A/C were used for normalisation. C. qRT-PCR analysis of Esrp1, Oct4, Nanog, Sox2, and c-Myc mRNA in Scr and Esrp1-depleted ES cells shows that there was a increase in their expression. RQ is relative quantity (n = 4). D. Representative Western blot analysis of Oct4, Nanog, Sox2 and c-Myc expression in nuclear extracts from Scr and Esrp1-depleted ES cells. Densitometric analysis of the Western blots is shown; A.U. is arbitrary unit (n = 3). doi:10.1371/journal.pone.0072300.g002 assays are also described in File S1. For rescue experiments, site directed mutagenesis was perfomed on pIBX-C-FF-EmGFP-B-ESRP1-2A (Kind gift of Pr. Russ Carstens) using QuikChange Site-Directed Mutagenesis Kit and following the manufacturer's protocol (Stratagene). Primers used are described in Table S2. pIBX-C-FF-EmGFP (Kind gift of Pr. Russ Carstens) was used as control. Reverse transfection with lipofectamine 2000 was used for delivery plasmid DNA into ES cells (Invitrogen). See File S1 for further details.

Teratoma Formation
Animals were bred in the central animal facility of the Molecular Biotechnology Center, University of Turin and were allowed free access to chow and drinking water and maintained under specific pathogen-free (SPF) conditions. Three hundred and fifty thousand Scramble (Scr) control or Esrp1-depleted ES cells were injected subcutaneously in recipient NOD-SCID-gamma (NSG) female mice. Teratomas were allowed to grow for 3 to 5 weeks. Mice were sacrificed using carbon dioxide euthanasia, followed by cervical dislocation to ameliorate suffering and teratomas were taken. Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin. For teratoma Role of Esrp1 in Stem Cells PLOS ONE | www.plosone.org volume assessment, 5610 4 cells were injected as described above. Tumor volume was measured with a calliper, using the formula: K (length6width6height).

Ethics Statement
All animal procedures were carried out under animal bioethics permit 116/92 (decreto legislativo 116/92) issued on the 11 th of September 2011 by the Bioethical committee of the University of Turin, Italy.

RNA-immunuprecipitation
ES cells (Scr and Esrp1-depleted) were lysed and cytoplasmic extracts used for immunoprecipitation with anti-ESRP1 antibody or rabbit IgG and for RNA extraction as described in File S1.
Protein was extracted using acetone/trichloroacetic acid precipitation. See File S1 for additional information.

Western Blotting
Protein was extracted using TENT buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton-X100) and a cocktail of protease inhibitors (Roche) and separated by SDS-PAGE. Fractionation of nuclear and cytoplasmic proteins is described in File S1. Antibodies used are described in Table S3. Cell culture and protein extraction for analysis of human ESRP1 expression in CD133+ kidney progenitor cells (KPC) [26] and kidney cancer stem cells (KCSC) [27] were generously provided by B. Bussolati and are described elsewhere. Densitometric analysis was performed using the volume analysis option of Quantity One software (Biorad Laboratories Inc).

Reporter Assay
ES cells were transfected with firefly luciferase reporter plasmids (Pou5f1-luc [28], pGL3-CMV-SOX2-59UTR [29] or Basic-luc control plasmid and control Renilla luciferase using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were processed using a Dual-Luciferase Reporter Assay System (Promega) and reactions were read on a luminometer (Promega). Reporter firefly luciferase values were normalized to those of the control Renilla to evaluate transfection efficiencies.

Knockdown of Esrp1 Cells Results in Enhanced Selfrenewal in Mouse ES Cells
To identify new RBPs that may affect self-renewal in pluripotent stem cells, we first selected a cluster of genes significantly down-regulated upon the onset of differentiation in mouse germline cell-derived pluripotent stem cells (GPSC), from our previously generated cDNA microarray data [22,25]. Only those genes that display an expression profile strongly similar to key pluripotency factors in both human and mouse ES cells were selected among them, using a previously described stem-cell specific CCN [19]. As expected, the resulting short-list (Table S4) was strongly enriched for proteins well known to play a critical role in ES cell fate determination, such as SALL4, OCT4, DPPA4 and L1TD1 [1,15,30,31]. Interestingly, this list also contained three RNA-binding proteins, the role of which has not been previously characterized in self-renewal of pluripotent cells: ELAV2, RBPMS2 and ESRP1. We decided to concentrate our functional validation on the latter, also considering that its expression has been strongly correlated to the expression of pluripotencyassociated factors such as L1TD1, DNMT3B, LIN28 and TDGF1 by previous independent studies [15].
To examine the function of ESRP1 in mouse ES cells, we knocked down the endogenous ESRP1 expression. ShRNAmediated depletion of ESRP1 by lentivirus in ES cells under basal conditions was confirmed both at the mRNA level, by qRT-PCR, and at the protein level, by western blotting and immunofluorescence analyses ( Figure 1A-C).
The conserved coexpression of Esrp1 with the main pluripotency genes and its down-modulation during pluripotent cells differentiation suggested that it could be a positive regulator of pluripotency. However, in contrast with this prediction, methylene blue and alkaline phosphatase (ALP) staining showed that Esrp1depleted ES cells formed undifferentiated colonies with greater efficiency than controls, both in the presence and in the absence of LIF. Scr ES cells differentiated in the absence of LIF and gave reduced ALP positivity. These results suggest that a reduction in ESRP1 may support LIF-independent ES cell propagation ( Figure 1D). Accordingly, Esrp1-depleted cells proliferated at higher rate than Scr controls ( Figure 1E). A second, independent ShRNA against Esrp1 gave similar results ( Figure S1). On inactivated mouse embryonic fibroblasts (Mefs), Esrp1-depleted ES cell colonies were morphologically similar to controls and stained positive for OCT4 and NANOG ( Figure S2).
As ESRP1 is well known for its involvement in maintaining an epithelial phenotype, we analysed the EMT status of the ES cells depleted for ESRP1 ( Figure 1F). There was a statistically significant increase in the FGFR2 IIIc/IIIb ratio in these cells compared to Scr controls. This was accompanied by a switch from the CD44v to CD44s isoform upon knockdown of ESRP1, as well as an increase in Slug, Snai1 and Vimentin which are known markers of EMT. These changes were confirmed with another ShRNA against ESRP1 ( Figure S1). On the other hand, there was a slight but significant decrease in E-cadherin expression in the ESRP1-depleted ES cells ( Figure 1F).
Confocal microscopy analysis of E14 ES cells revealed that ESRP1 was localized both in the nucleus and in the cytoplasm. Spermatogonial stem cells (SSCs) were characterised by qRT-PCR analysis. RQ is relative quantity (n = 4). B. Western blot analysis shows that ESRP1 is expressed in ES cells, in GPSCs as well as in SSCs. ES cells and GPSCs were lysed after 30 minutes' preplating to remove Mefs, while SSCs were scraped off together with the Mef-feeder layer for lysis. The primordial germ cell marker, MVH was enriched in SSCs versus the other cell lines. Actin was used for normalisation (n = 2). 3T3 fibroblasts were used as negative control. C. Confocal microcroscopy analysis revealed that ESRP1 is both nuclear and cytoplasmic in the SSCs as well. MVH staining was used as positive control. Scale bar is 5 mm. doi:10.1371/journal.pone.0072300.g005 Fractionation of nuclear and cytoplasmic proteins showed that ESRP1 was mainly located in the cytoplasm in the E14 ES cells (Figure 2A and B), in good agreement with previous reports on LS180 colon carcinoma cells [32]. Analysis of another ES cell line, v6.5, confirmed these results ( Figure S3). This finding, together with the fact that ESRP1 was found to bind to c-Myc and regulate its expression, prompted us to investigate whether ESRP1 could be involved in the regulation of pluripotency-related genes as well. We thus measured the levels of the core pluripotency factors, Oct4, Nanog and Sox2 as well as the Esrp1 target c-Myc, (Figure 2C and D) and found that they were higher, albeit moderately, both at mRNA and protein levels with respect to Scr controls [32].
In order to exclude the possibility of off-target effects of the ShRNAs, rescue experiments were performed using a mutant Esrp1. The four mutations inserted in the ShRNA binding site in pIBX-C-FF-EmGFP-B-ESRP1-2A plasmid ( Figure 3A) did not alter ESRP1 expression when compared to a plasmid expressing the wild-type ESRP1 ( Figure S4A) [33]. Importantly, transient introduction of this mutant plasmid into Esrp1-depleted ES cells reverted the phenotype observed after Esrp1 depletion. In particular, compared to cells transfected with the empty vector only, less ALP-positive colonies formed from Esrp1-depleted ES cells transfected with the ShRNA-immune Esrp1 (Figures 3B).
Restoration of Esrp1 expression also induced a decrease in FGFR2 IIIc/IIIb ratio and in core pluripotency genes expression ( Figure 3C and D, respectively) to a level that was comparable to that of Scr ES cells. Similar results were obtained with ES cells in a different genetic background (v6.5) ( Figure S4B) or after knockdown with a second ShRNA against Esrp1 ( Figure S4C). Taken together, these results indicate that Esrp1 was acting on self-renewal by regulating the expression of pluripotency transcription factors.

Depletion of Esrp1 Enhances Induced Pluripotent Stem (iPS) Cell Colony Generation
To establish whether, besides to its role in ES cell proliferation and self renewal, ESRP1 may also play a role in pluripotency acquisition, its expression was analysed during the reprogramming of primary Mefs into iPS cells as previously described [34]. In accordance with previous studies, the expression of Esrp1 shows a trend similar to the core pluripotency factors starting at Day 8 of reprogramming and increasing progressively ( Figure S5A) [35]. Moreover, depletion of Esrp1 in Mefs before the reprogramming procedure resulted in increased number of iPS cell colonies as evidenced by CDy1 and OCT4 staining compared to Scr controls or controls not infected with ShRNA-harbouring lentiviruses (NT) in agreement with the data on ES cells ( Figure S5B and C, respectively) [36]. EBs generated from iPS resulting from the three conditions expressed markers of the three germ layers upon EBs formation ( Figure S6A) and formed teratomas when injected into immuno-compromised mice ( Figure S6B).

Downregulation of Esrp1 in ES Cells Results in Impaired Early Differentiation Capacity
To assess the impact of Esrp1 depletion on the differentiation capacity of ES cells, we generated EBs from Scr control and Esrp1-depleted ES cells. At Day 5 of differentiation in vitro, Esrp1depleted ES cells showed impaired early differentiation, as indicated by the reduced levels of mesoderm (Brachyury) and endoderm (FoxA1) markers ( Figure 4A). Ectoderm (Fgf5) marker expression was comparable ( Figure 4A). In order to exclude the possibility that a reduction of ESRP1 expression might lead to trophectoderm differentiation of ES cells (as previously observed upon Oct4 and Sall4 depletion in ES cells [2,30]), we also analysed the expression of trophectoderm (Cdx2) marker. A reduction in Cdx2 expression was observed in Esrp1-depleted ES cells compared to Scr ES cells ( Figure 4A). Moreover, in the EBs derived from Esrp1-depleted ES cells, the expression of pluripotency-related genes remained elevated till Day 5 of differentiation and then become comparable to Scr controls by Day 8 ( Figure 4B). In order to test whether the delay in differentiation was temporal or absolute, in vivo teratoma assay was performed by injecting 3.5610 5 Esrp1-depleted cells in NOD-SCID-Gamma (NSG) mice [37]. Resulting teratomas in 8 out of 8 mice per group comprised all the three germ layers showing that these cells are indeed pluripotent and that their differentiation defect is only temporal ( Figure 4C). These data establish that the depletion of Esrp1 does not impair the multilineage differentiation potential of ES cells on the long term. Interestingly, the same assay, performed under nonsaturating conditions (5610 4 ES cells) showed that Esrp1 KD ES cell-derived teratomas grew significantly faster that those derived from Scr control ( Figure 4C). This was due to the higher proliferation rate, as evidenced by PCNA staining, of the more prominent neuroepithelium (asterisks and insets in Figure S7) in teratomas generated from Esrp1-depleted ES cells compared to Scr ES cells ( Figure S7).

ESRP1 is Expressed in Epithelial Stem Cells
In order to assess the applicability of these findings to other types of stem cells, we analysed the expression of ESRP1 in the unipotent spermatogonial stem cells or SSCs. As expected, the mouse SSCs express pluripotency markers like Oct4, Sox2, Klf4, Lin28, c-Myc as well as e-cadherin, but not Nanog compared to the pluripotent stem cells, ES and GPSCs [38,39]. ESRP1 is expressed both at the RNA and protein levels in SSCs ( Figure 5A and B). Interestingly, ESRP1 is present both in the nucleus and the cytoplasm of these cells, in accordance with the results obtained on ES cells ( Figure 5C and Figure 2A, respectively). Moreover, ESRP1 is also highly expressed in CD133+ human kidney progenitor cells, but downregulated in kidney cancer stem cells (protein extracts kindly provided by B. Bussolati) ( Figure S8) [26,27]. ESRP1 Binds to Pluripotency-related mRNAs As a protein containing RNA-recognition motifs, ESRP1 has been reported to associate with multiple mRNAs at their 59UTRs in the cytoplasm and to control their turnover and/or translational regulation [32]. To further analyse if pluripotency-related mRNAs are also bound by ESRP1, we performed RNA-immunoprecipitation with an anti-ESRP1 antibody on cytoplasmic extracts from mouse control ES cells. In agreement with the mentioned study, c-Myc mRNA was specifically immunoprecipitated in this assay, as no binding to the preimmune IgG ( Figure S9) or in Mefs (Table  S5) used as negative controls was detected ( Figure 6A). Most interestingly, Oct4 and Sox2 mRNAs were consistently co-immunoprecipitated with ESRP1( Figure S9). Accordingly, when ESRP1-depleted ES cells were used, there was statistically significant reduction in these pluripotency-related mRNAs, but not in Nanog or Lin28 mRNA, in the ESRP1 immunoprecipitation compared to the Scr controls ( Figure 6A). We further investigated whether ESRP1 could regulate the expression of Oct4 and Sox2 mRNAs through their 59UTR regions, by analysing translation of firefly luciferase reporter using pGL3-pCMV-59UTR-Oct4 and pGL3-pCMV-59UTR-Sox2 versus basic pGL3 vector. The luciferase reporter assays show that there was a statistically significant increase in luciferase activity when ESRP1 was depleted in ES cells compared to Scr controls ( Figure 6B), confirming that ESRP1 could inhibit the expression of Oct4 and Sox2 by binding their 59UTR regions.

Depletion of ESRP1 does not Affect the Decay Rate but Stimulates the Polysomal Loading of Pluripotencyrelated mRNAs
Since RBPs can regulate mRNA turnover, we investigated whether ESRP1 can influence the stability of pluripotency-related mRNAs. Actinomycin D-treated Scr and Esrp1-depleted ES cells showed similar mRNA decay rates of Oct4, Nanog, Sox2 and c-Myc, while as expected, there was a significant difference the Esrp1 mRNA degradation rate in these two cell types ( Figure S10).
Thus, at least in our hands, ESRP1 does not affect the analysed pluripotency-related mRNA stability in mouse ES cells.
It is generally believed that mRNAs actively being translated are associated with polysomes and that an increased polysome association indicates an increase in translation efficiency [40]. Based on this assumption, to provide further evidence supporting the inhibitory role of Esrp1 on mRNA translation, we performed polysomal profiling [32]. Cytoplasmic extracts from Scr and Esrp1-depleted ES cells were subjected to sucrose gradient fractionation and total RNA was extracted from polysomal fractions. While there was no difference in the ISCO fractionator-generated polysome profile of Scr and Esrp1-depleted ES cells ( Figure 7A), the amount of core pluripotency-related factors and c-Myc mRNAs in the pooled polysomal fractions was significantly higher upon depletion of Esrp1 compared to controls ( Figure 7A). These results suggest that the increased protein levels of pluripotency factors shown in Figure 2D are at least partially due to their increased translation. On the other hand, Nanog and Lin28 mRNA loading did not significantly differ between Scr and Esrp1-depleted ES cells ( Figure 7A). To further investigate in which sucrose gradient fractions ESRP1 was found in ES cells under basal conditions, we extracted proteins from the different fractions. The results show that ESRP1 is not located in the polysomal fractions ( Figure 7B), suggesting that ESRP1 may sequester pluripotency-related mRNAs away from the polysomes. Taken together, our findings indicate that depletion of ESRP1 in ES cells resulted in alteration in the level of expression of core transcription factors and the resultant cumulative effect of these changes affects early differentiation of these cells.

Discussion
ESRP1 has been shown to affect the mRNA translation of several cancer-related genes, including c-Myc and Fos, through direct binding of their 59 untranslated regions (UTRs) [20,32]. Moreover, Warzecha et al. demonstrated that ESRP1 can promote the alternative splicing of transcripts that switch splicing during epithelial to mesenchymal transition (EMT) [33,41,42]. Its role in EMT and tumor progression is getting increasingly studied [43,44,45].
In this study, we identified the RBP ESRP1 as a candidate regulator of self renewal and pluripotency in ES cells on the basis of its conserved co-expression with well-established pluripotency factors. Moreover, and most importantly, we provide strong functional evidence in support of this hypothesis, showing that ESRP1 controls the translation of pluripotency-related mRNAs in mouse ES cells and that its downregulation favours reprogramming of differentiated fibroblasts into pluripotent cells.
The capability of ESRP1 to restrain self renewal could appear paradoxical if its expression profile is considered. Indeed, ESRP1 is downregulated with the other pluripotency factors when both mouse GPSC and ES cells lose pluripotency, and it is upregulated when fibroblasts are reprogrammed into iPS cells. However, the experiments that we performed clearly indicate that ESRP1 functionally counteracts the activity of pluripotency-related genes. Indeed, reduced levels of Esrp1 enhances ES cells proliferation and self renewal, as shown by the higher number of undifferentiated colonies generated in vitro and by the increased size of teratoma obtained upon injecting non-saturating amounts of cells into immune-compromised animals compared to Scr controls. In addition, Esrp1 knockdown increases the number of iPS cell colonies obtained from primary differentiated fibroblasts. An alternative explanation for these phenotypes could be that a reduction of Esrp1 could transform pluripotent cells, a conceivable scenario especially if considering the previous implication of human ESRP1 as a tumor suppressor gene and in EMT [32,33]. However, our data seem to exclude this possibility. Indeed, Esrp1depleted cells are still capable of responding to differentiative stimuli, albeit with a slower kinetic when compared to control cells. Moreover, teratoma formation assays showed that these cells are still capable of giving rise to derivatives of the three germ layers. On the basis of these data, it seems safe to us to conclude that, in pluripotent cells, ESRP1 is a physiological regulator of the finely tuned balance between self renewal and commitment to a restricted developmental fate.
The simplest explanation to reconcile the functional properties of Esrp1 with its expression profile could be that the same transcriptional program that sustains the expression of pluripotency factors is responsible for the activation of a negative control layer, composed of Esrp1, and possibly, by other coding or noncoding genes. The existence of such negative fine-tuning would be very helpful to explain how the pluripotency machinery is kept under strict control, if considering that it is maintained in pluripotent cells by strong positive feedback loops between the core transcription factors [46]. An important next step to further support this scenario will be to investigate how Esrp1 is regulated at the transcriptional level. Interestingly, CHIP-seq data indicated that its promoter contains binding sites for both polycomb repressive complex (PRC) members (PRC2: EZH2 and SUZ12 and PRC1: RING1B) as well as for pluripotency-related genes (NANOG, KLF4, TCF3), suggesting that ESRP1 level in mouse ES cells is controlled by both pluripotency factors as well as PRC members [46,47]. However, further studies are needed to assess how important these factors are for the regulation of ESRP1 in ES cells under basal conditions and in response to differentiative stimuli.
Another important question is how does ESRP1 work at the molecular level? The increased levels of pluripotency factors that we detected in Esrp1-depleted cells as well as the luciferase reporter assays, together with the previous information on ESRP1 functions, strongly suggest that it may influence the expression of these genes by directly binding to their mRNA. Consistently, we found that the mRNA of c-Myc as well as the mRNA of Oct4, and Sox2 can be specifically co-immunoprecipitated with ESRP1. This interaction does not affect the decay rate of the studied mRNAs ( Figure S7), suggesting that ESRP1 probably does not bind to the 39UTR of these mRNAs and does not affect mRNA stability [48]. The alternative possibility that ESRP1 may primarily regulate translation was already supported by previous evidence. Indeed, it has been shown that ectopic expression of ESRP1 results in moderate changes in polysomal loading of a number of genes and that it can exert a differential effect on reporter RNA translation mediated by various 59UTRs [32]. The degree of 59UTRmediated translational inhibition by ESRP1 is strongly dependent on the complexity of the secondary structure of the 59UTRs. Esrp1 was also shown to control the translation of the oncogene and pluripotency factor c-Myc [32]. Consistent with this hypothesis, we found that, in Esrp1-depleted cells, the polysomal loading of pluripotency genes is significantly increased and that ESRP1 is not associated with polysomes. These results strongly support a scenario in which ESRP1 reduces the levels of pluripotency factors primarily by binding to their mRNAs and thus preventing their association to active ribosomes. As the pluripotency transcription factors are known to form part of a feed forward auto-regulatory loop, even the moderate rise in their protein levels may result in increased transcription as observed upon ESRP1 depletion in mouse ES cells (Figure 8) [46]. However, we cannot rule out the possibility that ESRP1 may prevent translation initiation, like for e.g., the physical binding of Esrp1 to these mRNAs may interfere with the binding of certain elongation initiation factors that form the initiation complex. We also cannot exclude that ESRP1 is acting, in parallel, on alternative splicing of other factors of the extended transcriptional network and thus influencing the self-renewing capacity of ES cells.

Conclusion
Our results strongly support the role of ESRP1 as a physiological regulator of pluripotency-related factors in mouse ES cells. The expression of ESRP1 in other stem cells, of both mouse and human origin, pinpoints the importance of this RBP in stem cell biology. Western blot analysis showing expression of mutated ESRP1-GFP compared to wild type ESRP1-GFP and empty vector using anti-GFP antibody. B. qRT-PCR analysis of the FGFR2 IIIc/IIIb ratio upon rescue in Esrp1-depleted v6.5 ES cells. Cells were transfected either with the empty vector (pEm) or with the mutated Esrp1 (Esrp1*). RQ is relative quantity. C. Rescue experiment was performed on ESRP1-depleted (E4) and control Scr E14 ES cells. E4 is another ShRNA wich gave efficient reduction of ESRP1 expression. qRT-PCR analysis shows the reduction in FGFR2 IIIc/IIIb ratio upon introduction of mutated Esrp1 (Esrp1*) in E4 cells. RQ is relative quantity (n = 3). (TIF)