BORIS/CTCFL is an RNA-binding protein that associates with polysomes
- Babatunji W Ogunkolade†1,
- Tania A Jones†1,
- Johan Aarum†1,
- Jaroslaw Szary1,
- Nicholas Owen1,
- Diego Ottaviani1,
- Muhammad A Mumin1,
- Shyam Patel1,
- Christopher A Pieri1,
- Andrew R Silver2 and
- Denise Sheer1Email author
© Ogunkolade et al.; licensee BioMed Central Ltd. 2013
Received: 15 July 2013
Accepted: 19 November 2013
Published: 26 November 2013
BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein.
We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.
We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.
KeywordsCTCF Ribosomes Nucleoli WNT signalling
CTCF is a highly conserved and ubiquitous protein that has widespread functions in transcription regulation and chromatin architecture. It acts as a silencing and activating transcriptional factor, a chromatin insulator and a mediator of chromatin looping, and is essential for life [1, 2]. Binding of CTCF to DNA is achieved primarily through its 11-zinc finger domain, which also facilitates protein-protein interactions [3–5]. CTCFL or BORIS (Brother of the Regulator of Imprinted Sites), is a paralogue of CTCF . BORIS has almost identical 11 zinc finger domains to CTCF, and the proteins are thought to have evolved during vertebrate development from a gene duplication event . However, the flanking N- and C-terminal regions of BORIS show no homology with CTCF or any other proteins . BORIS also lacks the modular substrates for specific post-translational modifications that are critical for CTCF function, suggesting divergent roles for the two proteins. Indeed, BORIS and CTCF are expressed in a mutually exclusive manner during male germ-line development, suggesting that BORIS is involved in reprogramming the paternal DNA-methylation patterns . Several lines of evidence suggest that BORIS plays a role in epigenetic regulation of gene expression. In tumour cell lines, where CTCF silences genes by DNA methylation, it has been shown that expression of BORIS can displace CTCF at these genes leading to local demethylation and gene activation [9–12]. Further epigenetic regulation is suggested by the binding of BORIS to the upstream binding factor (UBF), a transactivator of RNA polymerase I, which is involved in the maintenance of chromatin structure .
BORIS protein is readily detected in most cells and tissues , with abnormally high expression levels reported in several tumours and cell lines [15–22]. In contrast to previous findings suggesting divergence in the roles of BORIS and CTCF, recent evidence has shown that both proteins are able to mediate similar growth and tumour suppressor functions and both provide a protective effect during apoptosis . This finding warrants further characterisation of the functional properties of BORIS.
We previously showed that BORIS is present both in the cytoplasm and nucleus, and is enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism . The role of BORIS within the cytoplasm, which represents the major pool of BORIS protein in testis, has not been fully explored . Here, we hypothesized that cytoplasmic BORIS interacts with RNA, as shown for certain other Zn-finger proteins [25, 26], due to the subnuclear localisation of BORIS to the nucleolus, which is associated with RNA metabolism. To test this, we examined whether BORIS binds RNA and if so, whether this property changes in cells as they undergo phenotypic alterations. We show BORIS binds to distinct sets of RNA transcripts in neural stem cells and neurons and to a substantial amount of non-coding RNA. The transcripts are enriched for components of certain key cellular pathways including the WNT pathway. We further find that BORIS is associated with actively translating ribosomes. Together, our data suggest new roles for BORIS in the regulation of gene expression.
BORIS is an RNA binding protein
We therefore extended our investigation to determine whether BORIS interacts with RNA in other cell types and, if so, whether the interaction changes as cells undergo phenotypic alterations. We previously showed that BORIS is present at similar levels in hNP1 neural progenitor cells (Aruna Biomedical) and young neurons derived from hNP1 using well-defined culture conditions . Gene expression arrays confirmed no significant change in expression of BORIS during neural differentiation (data available at NCBI’s Gene Expression Omnibus , accession number GSE42294). Expression of BORIS in hNP1 and HEK293T cells was confirmed by partial sequencing of PCR product (Additional file 2: Figure S2).
Identification of poly(A) RNAs bound to BORIS
To identify which transcripts were associated with BORIS in hNP1 and 6dN cells we immunoprecipitated the protein from cellular extracts. We then isolated the RNA and converted it to cDNA, which was hybridized to gene expression arrays. The signals from the arrays were then compared to those obtained from total RNA isolated from hNP1 and 6dN cells. Transcripts were scored as associated with BORIS if the fold change was larger than two and the p-value (ANOVA) was less than 0.01 (see Methods). In total, we identified 1097 and 962 probes representing 863 and 771 unique transcripts associated with BORIS in hNP1 and 6dN cells, respectively (Additional file 3: Table S1 and Additional file 4: Table S2). Of these, 88 transcripts were common to both hNP1 cells and 6dN cells (Figure 4C). These findings were confirmed for several genes by the validation of enrichment using RT-qPCR in hNP1 and 6dN cells (Figure 4D,E). In addition, we showed that the association of transcripts with BORIS did not correlate with their up- or down regulation during neural differentiation (Additional file 5: Table S3).
Characterisation of BORIS-bound transcripts
p- values for PANTHER analysis of pathways, molecular function and biological processes of transcripts bound in (A) hNP1 and (B) hNP1 cells differentiated to neurons over 6 days (6dN)
BORIS bound transcripts in hNP1
WNT signaling pathway
Cadherin signaling pathway
nucleic acid binding
transcription factor activity
transcription regulator activity
RNA splicing factor activity, transesterification mechanism
RNA helicase activity
nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
primary metabolic process
cellular component organization
intracellular protein transport
establishment or maintenance of chromatin architecture
nervous system development
BORIS bound transcripts in 6dN
WNT signaling pathway
Inflammation mediated by chemokine and cytokine signaling pathway
Alzheimer disease-presenilin pathway
TGF-beta signaling pathway
nucleic acid binding
transferase activity, transferring glycosyl groups
ubiquitin-protein ligase activity
RNA splicing factor activity, transesterification mechanism
primary metabolic process
nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
intracellular protein transport
protein metabolic process
PANTHER  was then used for functional analysis of translated protein products for BORIS-associated transcripts. Significant enrichment (p < 0.05) was found in DNA and RNA-binding proteins, as well as RNA splicing factor activity in both hNP1 and 6dN cells (Figure 5C and D and Table 1). PANTHER analysis also showed that BORIS-associated transcripts are involved in diverse biological processes (Figure 5E and F and Table 1). Over-represented biological processes for transcripts from hNP1 include metabolic process, cellular component organization, protein transport, organelle organization, and nervous system development. Over-represented biological processes for transcripts from 6dN include cell cycle, primary metabolic process, cellular process, transport (protein, ion etc.) and mitosis.
BORIS expression activates the β-catenin dependent WNT canonical pathway
To determine directly if BORIS influences the activation of the WNT pathway, we then used a luciferase reporter assay where the luciferase expression is driven by tandem repeats of multiple copies of the consensus TCF/LEF- β-catenin responsive element (SABiosciences). LiCl, an inhibitor of GSK-3, was used as a positive control for pathway activation . Transient over-expression of BORIS in HEK293T cells led to a more than four-fold increase in luciferase activity compared to cells transfected with empty vector alone (Figure 6C). This activation was dependent on β-catenin as siRNA knock-down of β-catenin caused a significant reduction in the effect of BORIS over-expression in the TCF/LEF luciferase assay (Figure 6D, E and F).
BORIS associates with polysomes
Polysome profiling of HEK293T cells showed a similar sedimentation profile of BORIS to that observed in hNP1 and 6dN cells (Figure 7B). Inhibition of translation in HEK293T cells using puromycin, which causes premature chain termination and polysomal dissociation  shifted BORIS and RPL7 to the first, light fractions (Figure 7B). Furthermore, both RNase A digestion and dissociation of ribosomes into subunits by 30 mM EDTA with the concomitant release of mRNA and the 5S ribosomal protein [34, 35], also shifted the sedimentation of BORIS and to a lesser extent RPL7 towards lighter fractions (Figure 7B). Together, these findings suggest that BORIS associates with actively translating ribosomes in these cells.
Here, we provide evidence that BORIS, best known for its role in DNA binding and transcriptional regulation, also binds RNA in vitro and associates with subsets of mRNAs and with translating ribosomes in neural stem cells and young neurons. The ability to bind to both DNA and RNA is not unique to BORIS, and is a feature of certain other zinc finger containing proteins [36–41]. The zinc finger domains of BORIS, with which it associates with DNA, are almost identical to those in CTCF and the proteins are reported to share DNA binding sites in the genome [8, 11, 24]. A recent study has suggested that the zinc fingers in BORIS are needed for both nuclear and nucleolar localisation . It remains to be established whether the zinc finger motifs are important for the RNA-binding properties of BORIS, as is the case for TFIIIA , WT1  and certain other proteins .
An interesting feature of BORIS is that its mRNA expression is extremely low in cultured or primary cells, yet the protein levels are readily detectable. This is consistent, however, with a report that RNA binding proteins tend to exhibit high protein stability and translational efficiency, yet their transcripts have a short half-life . The authors of the report suggest that tight regulation of the levels of RNA binding proteins is required since a significant change in their expression may affect many targets altering global expression levels.
Although the majority of BORIS-associated transcripts differ between hNP1 and 6dN cells, similarities are observed in the pathways in which the transcripts are involved in the two cell types. For example, BORIS-associated transcripts in both cell types encode proteins involved in the canonical WNT pathway. WNT signalling is crucial in the regulation of a wide range of cellular processes such as apoptosis, cell proliferation, and differentiation, including that of neural stem cells [46–48].
A role for BORIS in regulating WNT signalling is supported by our finding that BORIS increases the activity of a TCF/LEF reporter following transient over-expression in HEK293T cells (Figure 6C). As the reporter activation is dependent on β-catenin (Figure 6F), BORIS is unlikely to affect the TCF/LEF reporter directly, but rather to have a post-transcriptional role. BORIS associates with several transcripts coding for regulatory components of the pathway and it is therefore conceivable that its over-expression may affect the translation of WNT pathway components. Indeed, BORIS over-expression leads to increased TCF3 and WNT5A protein levels, whilst their respective transcript levels are decreased (Figure 6A and B) Although there are several possible explanations for this increase in protein levels, for example post-translational modifications leading to greater protein stability [49, 50], the fact that BORIS associates to these transcripts as well as to actively translating ribosomes (Figure 7A,B) argues for a translational effect of BORIS on these proteins. However, further studies are required to conclusively answer this question.
The biological consequences of the association of BORIS with different transcripts within individual pathways in hNP1 and 6dN cells have yet to be determined. BORIS may be involved in coordinated regulation of different transcripts within certain pathways at specific time points of cell development or differentiation.
We show that BORIS can directly interact with RNA in vitro and is associated with a subset of mRNA and translating ribosomes in neural stem cells and young neurons. Transient over-expression of BORIS increases the protein levels of several BORIS-associated transcripts without any concomitant increase in transcript levels suggesting a role for BORIS in translational control.
Human neural stem cells, hNP1, derived from the cell line WA09 (46,XX) (Aruna Biomedical, hNP7013.1), were cultured in Neurobasal medium (Invitrogen, 21103–049) supplemented with B27 (Invitrogen, 12587–010), FGF-2 10 ng/ml, 1% penicillin/streptomycin and 2 mM glutamine as previously reported . Half the medium was changed every other day. We induced differentiation by omitting FGF-2 from the medium as described by Shin et al., . Human embryonic kidney cells, HEK293T, were maintained in RPMI containing 10% fetal bovine serum 1% penicillin/streptomycin (Invitrogen) and 2 mM glutamine at 37°C in 5% CO2 (Invitrogen).
BORIS antibody ab18337 (1:1000 dilution, Abcam), CTCF antibody 07–729 (1:1000 dilution, Millipore) and GAPDH antibody 14C10 (1:1000 dilution, Cell Signaling) were used in Western data shown. The specificity of the BORIS antibody was determined using recognition of GFP-tagged recombinant BORIS and non-recognition of GFP-tagged recombinant CTCF protein by western blotting (Additional file 8: Figure S3). The specificity of the BORIS antibody has also previously been confirmed by siRNA knock down, peptide competition and the recognition of recombinant BORIS . WNT3a (C64F2) rabbit monoclonal antibody, WNT5a/b (C27E8) rabbit monoclonal antibody and LRP6 (C5C7) rabbit monoclonal antibody were from the WNT signaling antibody sampler kit, 2915 (Cell Signaling) and TCF3 (D15G11) rabbit monoclonal antibody and TCF4 (C48H11) rabbit monoclonal antibody were from the TCF/LEF1 antibody sampler kit, 9383 (Cell Signaling) and were used at 1:1000 dilution.
Run-on transcription assay
For immunodetection of newly synthesized RNA, HEK293T cells grown on coverslips were briefly incubated (15–20 minutes at 37°C) with 2 mM 5'-fluorouridine (5-FU) (F5130; Sigma) . Cells were then fixed with 4% paraformaldehyde for 10 min, permeabilised with 1% Triton X-100, and incorporation of 5-FU into nascent RNA was monitored using antibody against halogenated UTP (1:100, anti-bromodeoxyuridine (BrdU) clone BU-33, B8434; Sigma) and a Texas Red-conjugated secondary antibody. Nuclei were stained with 0.1 mg/ml 4’, 6-Diamidino-2-phenylindole (DAPI, Molecular Probes) and mounted in Mowiol (Calbiochem). For standard 2 dimensional analysis, specimens were visualized using a Zeiss Axiophot microscope equipped for epifluorescence using Zeiss plan-neofluar 100x objective. Separate grey-scale images were recorded with a cooled CCD-camera (Hamamatsu). Image analysis was performed using SmartCapture X software (Digital Scientific).
Identification of nuclear export signal
Identification of a putative nuclear export signal (NES) in the C terminal region was performed using NetNES (http://www.cbs.dtu.dk/services/NetNES/).
Oligo-dT precipitation of BORIS
Cells were trypsinised, washed in ice cold buffer A (10 mM Tris–HCl pH 7.5, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mM KCl) and lysed in buffer C (10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, and 2unit/μl RNaseOUT). 1000 μg of protein lysate was incubated with 100 μl oligo-dT-dynabeads (Invitrogen) and incubated at 4°C for 30 minutes. Oligo-dT/mMRNA/protein complex was separated from unbound proteins using an Invitrogen magnetic separator. The beads were washed five times with solution D (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.02% NP-40 and 1U/ml RNaseOUT) using at least twice the lysate volume for washing. Beads and attached complexes were re-suspended in 20–40 μl PAGE loading buffer for western blot analysis.
Identification of BORIS bound mRNAs
Immunoprecipitation of BORIS-mRNA complexes was used to assess the association of BORIS with target mRNAs as previously described with some modification . Briefly, 10–20 million cells were washed with PBS and lysed in ice cold swelling buffer A (25 mM HEPES, 1.5 mM MgCl2, 85 mM KCl, pH 8.0) for 5 minutes. After spinning for 5 minutes at 4°C, the pellet was lysed in buffer C (buffer A supplemented with 0.2% NP-40, 1% Triton X-100, with 0.01% saponin, 1 × protease inhibitor cocktail (Roche), 2 U/ml of RNaseOUT and phosphatase inhibitors mix (5 mM each of Sodium Fluoride, Sodium Orthovanadate and beta-glycerophosphate)) for 30 minutes and cleared by centrifugation at 21,000 g for 10 minutes. The cleared supernatant was incubated with 10 μg BORIS antibody (ab18337, Abcam) coupled to dynabead protein A (100 μl) (Invitrogen) for 1–2 hours at 4°C. After extensive washes with buffer D (buffer A, supplemented with phosphatase inhibitors mix, 1 × protease inhibitor cocktail (Roche), 0.1 U/ml of RNaseOut, 0.02% NP-40 and 0.25% Triton X-100), the bead-protein complex was incubated with 50 units of DNase 1 containing 100 units of RNaseOUT for 5 minutes at 37°C. An equal volume of proteinase K containing buffer was added and incubated for another 15 minutes at 37°C. RNA was extracted with standard phenol chloroform procedure and precipitated with 2 μl of glycogen (Sigma-Aldrich).
The RNA was used for either hybridization to Affymetrix U133 plus 2.0 expression arrays or for RT-qPCR verification of BORIS target transcripts. For array analysis, double stranded cDNA was synthesized from 1.5 – 5 μg total RNA using the Affymetrix One-cycle cDNA synthesis kit following the manufacturer’s instructions (Affymetrix). Synthesis of Biotin-labeled cRNA was performed using the Affymetrix GeneChip IVT labeling kit followed by purification with the sample cleanup module. Labeled cRNA was then fragmented and hybridized to Affymetrix GeneChip Human Genome U133Plus 2.0 arrays overnight. Hybridisation and scanning was performed in house at Barts Cancer Institute. For RT-qPCR analysis, RNA in the IP material was reverse-transcribed to cDNA using superscript III (Invitrogen) following the manufacturer’s instructions. Quantitative real time PCR was performed on ABI7500 equipment using gene-specific primer pairs and amplification condition of 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 secs at 95°C and 45 secs at 60°C.
Total RNA was isolated using silica-based spin-column extraction kit (RNeasy mini kit, Qiagen) following the manufacturer’s protocol. Total RNA was treated with RNase-free DNase1 (Ambion) to reduce genomic DNA contamination. RNA integrity was evaluated using the Agilent Bioanalyzer. Two micrograms of total RNA was reverse transcribed with SuperScriptase III (Invitrogen) using Oligo-dT primers or random hexamers according to the manufacturer’s protocol. Negative (-RT) controls contained RNase-free water substituted for reverse transcriptase.
Recombinant BORIS purification
The mammalian expression plasmid pM49-T4738 carries BORIS with an N-terminal HaloTag. Adherent HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) using standard methods. Cells were cultured for 48 h prior to harvest. Media were aspirated and cells washed in cold PBS before removal by cell scraping. Cells were centrifuged at 2000 × g for 5 min. The cell pellet containing over-expressed HaloTag-BORIS was stored at -80°C overnight. The cell pellet was lysed in lysis buffer (50 mM HEPES pH7.5, 150 mM NaCl) supplemented with BaculoGold protease inhibitor (BD Biosciences). HaloTag-BORIS was purified as per manufacturers protocol (Promega). The cell pellet was lysed on ice in 1 ml of lysis buffer per 2 × 107 cells for 10 minutes, followed by 5 min pulse sonication (30 seconds ON, 30 seconds OFF) using Diagenode's Bioruptor 3 min (high setting at 4°C). Crude lysate was centrifuged at 10,000 × g for 30 min. The resulting cleared lysate was mixed with 100 ml HaloLink resin (25% slurry), incubated for 1 h rotating, and washed three times with lysis buffer. Washes were removed through centrifugation of the HaloLink resin at 1000 ×g for 5 min and aspiration. At the final wash, the resin was resuspended in cleavage buffer (lysis buffer supplemented with 15 mg/ml TEV protease) and rotated for 2 h at room temperature. Resin was centrifuged at 2000 x g for 5 min and supernatant removed. TEV protease was removed by the addition of HisLink resin to the supernatant and incubation for 20 min rotating at room temperature. HisLink was removed through centrifugation at 1000 × g for 5 min and the resulting supernatant snap frozen in liquid nitrogen and stored at -80°C. Quantification of the protein was carried out using BCA Protein Assay (Thermo Scientific). Purification was confirmed through Western blot analysis using rabbit anti-BORIS antibody (Abcam ab18337).
Western blot analysis
Protein extracts or precipitated protein complexes were separated on a 4–12% gradient NuPAGE polyacrylamide gel (Invitrogen) and then blotted onto nitrocelluose membrane (Invitrogen) as described by Jones et al. . After incubation with blocking solution (Tris-buffered saline containing 5% skimmed milk and 0.1% Tween-20) the membrane was incubated with corresponding antibodies overnight at 4°C. After several washes, bands were revealed with the corresponding horseradish peroxidase coupled secondary antibody and detected using the ECL detection kit (GE Healthcare) according to the manufacturer’s protocol. Densitometry scanning of the intensity of bands on the Western blot was quantified using ImageJ. The p-values were obtained using one-way ANNOVA test after intensity values were normalised to GAPDH levels.
In vitro binding assay
For RNA and DNA binding assays, ~1 mg of purified BORIS protein was incubated with 125 nM of each biotinylated homopolymer in 400 ml of Binding Buffer (RBB, 150 mM NaCl, 20 mM Tris (pH7.5), 1 mM dithiothreitol [DTT] and 0.2% NP-40 at 4°C overnight. Nucleotide:protein complexes were isolated by addition of 20 ml prewashed Dynabeads M280 Streptavidin (Invitrogen) to the reaction for 30 min rotating at room temperature. Complexes were magnetically captured and washed three times in RBB. After the final wash, beads were resuspended in 10 ml NuPAGE LDS sample buffer supplemented with 5 mM DTT, heated to 70°C for 5 min. Captured proteins were resolved by 4 - 12% SDS/PAGE and analysed by Western blot using anti-BORIS antibody.
Analysis of microarray data
Affymetrix Expression array files were analysed using Partek ® software, version 6.5 Copyright © 1993–2010 (Partek Inc.). Principle component analysis (PCA) was applied to identify any independent sources of variation in the data. We compared data for BORIS bound RNA transcripts with genome-wide gene expression profiles for each selected cell type (hNP1, 6dN and HEK293T cells) with at least two biological replicates. A t-test was performed and transcripts were considered to be preferentially associated with BORIS when the signals from the immunoprecipitated RNA fractions were enriched more than 2 fold, with a p-value < 0.01. The gene expression data have been deposited in NCBI’s Gene Expression Omnibus  and are accessible through GEO series accession number GSE42294 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42294).
Pathway analysis and functional classification
We used Protein ANalysis THrough Evolutionary Relationships (PANTHER) software to identify significantly enriched functional pathways and Gene Ontology (GO) terms associated with BORIS-bound transcripts . Proteins were functionally classified using the PANTHER system (http://www.PANTHERdb.org).
Quantitative real-time PCR
List of primer pairs selected for RT-qPCR confirmation of BORIS associated transcripts in (A) hNP1 an hNP1 cells differentiated to neurons over 6 days (6dN), (B) HEK293T cells over-expressing BORIS
Preparation and analysis of polysomes
Cell extracts for polysome analysis were prepared as described by Camacho-Vanegas O et al. . Briefly, 5 x 108 cells were incubated with cyclohexemide for 30 minutes then washed with ice-cold PBS containing 100 μg/ml cycloheximide (Sigma) to block ribosomes at the step of elongation. Cells were lysed for 5 minutes in cold 1 x polysome buffer (10 mM Tris–HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2 and 0.05% NP40) containing 100 μg/ml cycloheximide. Cytoplasmic extracts were obtained after centrifugation at 10,000 × g for 5 min at 4°C, and then loaded onto a linear (10 – 50%) sucrose gradient in polysome buffer, and centrifuged at 100,000 × g for 2 h at 4°C. 650 μl fraction were collected and absorbance at 260 and 254 nm was measured using a spectrophotometer (Nanodrop). Aliquots of each fraction was mixed with 4 x PAGE loading buffer and analysed on a 4 – 12% NUPAGE gels.
Cloning and transfection
The GFP-BORIS, GFP-CTCF and pEGFP-C3 vectors were transfected into HEK293T cells using FuGene 6-HD (Roche) according to manufacturer’s protocol as previously described .
Activation of relative TCF/LEF-dual luciferase assay
The effect of BORIS on the WNT pathway was evaluated by measuring the activation of transcription factor TCF/LEF with the Cignal TCF/LEF reporter assay kit (SA Biosciences). In the first instance, HEK293T cells were cells co-transfected with TCF/LEF reporter constructs and either C3-BORIS or C3-empty vector, using Lipofectamin-2000 (Invitrogen) according to manufacturer's instructions. In other experiments, non-targeted or β-catenin siRNAs (Ambion) were combined with the C3 BORIS or C3 empty vector and co-transfected with TCF/LEF reporter constructs (SA Biosciences) according to manufacturer's instructions. The TCF/LEF reporter used a mixture of an inducible β-catenin-responsive luciferase construct and a constitutively expressing Renilla element (40:1). After 48 hrs incubation cells were collected and analyzed for TCF/LEF activity using a dual-luciferase assay kit (Promega-Biosciences). TCF/LEF activation values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in duplicate, and the standard deviations are indicated.
hNP1 cells differentiated over 6 days
- BORIS (CTCFL):
Brother of the Regulator of Imprinted Sites
Human neural progenitor cells
Ribosomal protein L7.
This work was supported by Cancer Research UK programme grant C5321/A8318. HEK293T cells were a kind gift from Professor Aine MacKnight. The authors have declared that no conflict of interest exists.
- Phillips JE, Corces VG: CTCF: Master Weaver of the Genome. Cell. 2009, 137 (7): 1194-1211. 10.1016/j.cell.2009.06.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Filippova GN, Qi CF, Ulmer JE, Moore JM, Ward MD, Hu YJ, Loukinov DI, Pugacheva EM, Klenova EM, Grundy PE: Tumor-associated zinc finger mutations in the CTCF transcription factor selectively alter tts DNA-binding specificity. Cancer Res. 2002, 62 (1): 48-52.PubMedGoogle Scholar
- Ohlsson R, Lobanenkov V, Klenova E: Does CTCF mediate between nuclear organization and gene expression?. Bioessays. 2010, 32 (1): 37-50. 10.1002/bies.200900118.View ArticlePubMedGoogle Scholar
- Filippova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, Goodwin G, Neiman PE, Collins SJ, Lobanenkov VV: An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol. 1996, 16 (6): 2802-2813.PubMed CentralView ArticlePubMedGoogle Scholar
- Zlatanova J, Caiafa P: CTCF and its protein partners: divide and rule?. J Cell Sci. 2009, 122 (Pt 9): 1275-1284.View ArticlePubMedGoogle Scholar
- Hore TA, Deakin JE, Marshall Graves JA: The evolution of epigenetic regulators CTCF and BORIS/CTCFL in amniotes. PLoS Genet. 2008, 4 (8): e1000169-10.1371/journal.pgen.1000169.PubMed CentralView ArticlePubMedGoogle Scholar
- Campbell AE, Martinez SR, Miranda JL: Molecular architecture of CTCFL. Biochem Biophys Res Commun. 2010, 396 (3): 648-650. 10.1016/j.bbrc.2010.04.146.View ArticlePubMedGoogle Scholar
- Klenova EM, Morse HC, Ohlsson R, Lobanenkov VV: The novel BORIS + CTCF gene family is uniquely involved in the epigenetics of normal biology and cancer. Semin Cancer Biol. 2002, 12 (5): 399-414. 10.1016/S1044-579X(02)00060-3.View ArticlePubMedGoogle Scholar
- Hong JA, Kang Y, Abdullaev Z, Flanagan PT, Pack SD, Fischette MR, Adnani MT, Loukinov DI, Vatolin S, Risinger JI: Reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter coincides with derepression of this cancer-testis gene in lung cancer cells. Cancer Res. 2005, 65 (17): 7763-7774.PubMedGoogle Scholar
- Ulaner GA, Vu TH, Li T, Hu JF, Yao XM, Yang Y, Gorlick R, Meyers P, Healey J, Ladanyi M: Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site. Hum Mol Genet. 2003, 12 (5): 535-549. 10.1093/hmg/ddg034.View ArticlePubMedGoogle Scholar
- Vatolin S, Abdullaev Z, Pack SD, Flanagan PT, Custer M, Loukinov DI, Pugacheva E, Hong JA, Morse H, Schrump DS: Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes. Cancer Res. 2005, 65 (17): 7751-7762.PubMedGoogle Scholar
- Kang Y, Hong JA, Chen GA, Nguyen DM, Schrump DS: Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells. Oncogene. 2007, 26 (30): 4394-4403. 10.1038/sj.onc.1210218.View ArticlePubMedGoogle Scholar
- van de Nobelen S, Rosa-Garrido M, Leers J, Heath H, Soochit W, Joosen L, Jonkers I, Demmers J, van der Reijden M, Torrano V: CTCF regulates the local epigenetic state of ribosomal DNA repeats. Epigenetics Chromatin. 2010, 3 (1): 19-10.1186/1756-8935-3-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Jones TA, Ogunkolade BW, Szary J, Aarum J, Mumin MA, Patel S, Pieri CA, Sheer D: Widespread Expression of BORIS/CTCFL in Normal and Cancer Cells. PLoS One. 2011, 6 (7): e22399-10.1371/journal.pone.0022399.PubMed CentralView ArticlePubMedGoogle Scholar
- D'Arcy V, Abdullaev ZK, Pore N, Docquier F, Torrano V, Chernukhin I, Smart M, Farrar D, Metodiev M, Fernandez N: The potential of BORIS detected in the leukocytes of breast cancer patients as an early marker of tumorigenesis. Clin Cancer Res. 2006, 12 (20 Pt 1): 5978-5986.View ArticlePubMedGoogle Scholar
- D'Arcy V, Pore N, Docquier F, Abdullaev ZK, Chernukhin I, Kita GX, Rai S, Smart M, Farrar D, Pack S: BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumours. Br J Cancer. 2008, 98 (3): 571-579. 10.1038/sj.bjc.6604181.PubMed CentralView ArticlePubMedGoogle Scholar
- Dougherty CJ, Ichim TE, Liu L, Reznik G, Min WP, Ghochikyan A, Agadjanyan MG, Reznik BN: Selective apoptosis of breast cancer cells by siRNA targeting of BORIS. Biochem Biophys Res Commun. 2008, 370 (1): 109-112. 10.1016/j.bbrc.2008.03.040.View ArticlePubMedGoogle Scholar
- Hoffmann MJ, Müller M, Engers R, Schulz WA: Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies. Biochem Pharmacol. 2006, 72 (11): 1577-1588. 10.1016/j.bcp.2006.06.020.View ArticlePubMedGoogle Scholar
- Kholmanskikh O, Loriot A, Brasseur F, De Plaen E, De Smet C: Expression of BORIS in melanoma: lack of association with MAGE-A1 activation. Int J Cancer. 2008, 122 (4): 777-784. 10.1002/ijc.23140.View ArticlePubMedGoogle Scholar
- Looijenga LH, Hersmus R, Gillis AJ, Pfundt R, Stoop HJ, van Gurp RJ, Veltman J, Beverloo HB, van Drunen E, van Kessel AG: Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer Res. 2006, 66 (1): 290-302. 10.1158/0008-5472.CAN-05-2936.View ArticlePubMedGoogle Scholar
- Risinger JI, Chandramouli GV, Maxwell GL, Custer M, Pack S, Loukinov D, Aprelikova O, Litzi T, Schrump DS, Murphy SK: Global expression analysis of cancer/testis genes in uterine cancers reveals a high incidence of BORIS expression. Clin Cancer Res. 2007, 13 (6): 1713-1719. 10.1158/1078-0432.CCR-05-2569.View ArticlePubMedGoogle Scholar
- Woloszynska-Read A, James SR, Link PA, Yu J, Odunsi K, Karpf AR: DNA methylation-dependent regulation of BORIS/CTCFL expression in ovarian cancer. Cancer Immun. 2007, 7: 21-PubMed CentralPubMedGoogle Scholar
- Tiffen JC, Bailey CG, Marshall AD, Metierre C, Feng Y, Wang Q, Watson SL, Holst J, Rasko JE: The cancer-testis antigen BORIS phenocopies the tumor suppressor CTCF in normal and neoplastic cells. Int J Cancer. 2013, 133 (7): 1603-1613. 10.1002/ijc.28184.View ArticlePubMedGoogle Scholar
- Loukinov DI, Pugacheva E, Vatolin S, Pack SD, Moon H, Chernukhin I, Mannan P, Larsson E, Kanduri C, Vostrov AA: BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc Natl Acad Sci U S A. 2002, 99 (10): 6806-6811. 10.1073/pnas.092123699.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown RS: Zinc finger proteins: getting a grip on RNA. Curr Opin Struct Biol. 2005, 15 (1): 94-98. 10.1016/j.sbi.2005.01.006.View ArticlePubMedGoogle Scholar
- Morrison AA, Viney RL, Ladomery MR: The post-transcriptional roles of WT1, a multifunctional zinc-finger protein. Biochim Biophys Acta. 2008, 1785 (1): 55-62.PubMedGoogle Scholar
- Edgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002, 30 (1): 207-210. 10.1093/nar/30.1.207.PubMed CentralView ArticlePubMedGoogle Scholar
- Fiorentino FP, Macaluso M, Miranda F, Montanari M, Russo A, Bagella L, Giordano A: CTCF and BORIS Regulate Rb2/p130 Gene Transcription: A Novel Mechanism and a New Paradigm for Understanding the Biology of Lung Cancer. Mol Cancer Res. 2011, 9 (2): 225-233. 10.1158/1541-7786.MCR-10-0493.View ArticlePubMedGoogle Scholar
- Renaud S, Loukinov D, Alberti L, Vostrov A, Kwon YW, Bosman FT, Lobanenkov V, Benhattar J: BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells. Nucleic Acids Res. 2011, 39 (3): 862-873. 10.1093/nar/gkq827.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen P, Cui H, Bisht KS, Sun L, Patel K, Lee RS, Kugoh H, Oshimura M, Feinberg AP, Gius D: CTCFL/BORIS is a methylation-independent DNA-binding protein that preferentially binds to the paternal H19 differentially methylated region. Cancer Res. 2008, 68 (14): 5546-5551. 10.1158/0008-5472.CAN-08-1005.PubMed CentralView ArticlePubMedGoogle Scholar
- Mi H, Lazareva-Ulitsky B, Loo R, Kejariwal A, Vandergriff J, Rabkin S, Guo N, Muruganujan A, Doremieux O, Campbell MJ: The PANTHER database of protein families, subfamilies, functions and pathways. Nucleic Acids Res. 2005, 33 (suppl 1): D284-D288.PubMed CentralPubMedGoogle Scholar
- Klein PS, Melton DA: A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci U S A. 1996, 93 (16): 8455-8459. 10.1073/pnas.93.16.8455.PubMed CentralView ArticlePubMedGoogle Scholar
- Unsworth H, Raguz S, Edwards HJ, Higgins CF, Yague E: mRNA escape from stress granule sequestration is dictated by localization to the endoplasmic reticulum. FASEB J. 2010, 24 (9): 3370-3380. 10.1096/fj.09-151142.View ArticlePubMedGoogle Scholar
- Nolan RD, Arnstein HR: The dissociation of rabbit reticulocyte ribosomes into subparticles active in protein synthesis. Eur J Biochem. 1969, 10 (1): 96-101.View ArticlePubMedGoogle Scholar
- Blobel G: Isolation of a 5S RNA-protein complex from mammalian ribosomes. Proc Natl Acad Sci U S A. 1971, 68 (8): 1881-1885. 10.1073/pnas.68.8.1881.PubMed CentralView ArticlePubMedGoogle Scholar
- Cassiday LA, Maher LJ: Having it both ways: transcription factors that bind DNA and RNA. Nucleic Acids Res. 2002, 30 (19): 4118-4126. 10.1093/nar/gkf512.PubMed CentralView ArticlePubMedGoogle Scholar
- Clemens KR, Wolf V, McBryant SJ, Zhang P, Liao X, Wright PE, Gottesfeld JM: Molecular basis for specific recognition of both RNA and DNA by a zinc finger protein. Science. 1993, 260 (5107): 530-533. 10.1126/science.8475383.View ArticlePubMedGoogle Scholar
- Hall TM: Multiple modes of RNA recognition by zinc finger proteins. Biochim Biophys Acta. 2005, 15 (3): 367-373.Google Scholar
- Ladomery M, Sommerville J: Binding of Y-box proteins to RNA: involvement of different protein domains. Nucleic Acids Res. 1994, 22 (25): 5582-5589. 10.1093/nar/22.25.5582.PubMed CentralView ArticlePubMedGoogle Scholar
- Scherrer T, Femmer C, Schiess R, Aebersold R, Gerber A: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins. Genome Biol. 2011, 12 (1): R3-10.1186/gb-2011-12-1-r3.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhai G, Iskandar M, Barilla K, Romaniuk PJ: Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1†. Biochemistry. 2001, 40 (7): 2032-2040. 10.1021/bi001941r.View ArticlePubMedGoogle Scholar
- Rosa-Garrido M, Ceballos L, Alonso-Lecue P, Abraira C, Delgado MD, Gandarillas A: A cell cycle role for the epigenetic factor CTCF-L/BORIS. PLoS One. 2012, 7 (6): e39371-10.1371/journal.pone.0039371.PubMed CentralView ArticlePubMedGoogle Scholar
- Caricasole A, Duarte A, Larsson SH, Hastie ND, Little M, Holmes G, Todorov I, Ward A: RNA binding by the Wilms tumor suppressor zinc finger proteins. Proc Natl Acad Sci U S A. 1996, 93 (15): 7562-7566. 10.1073/pnas.93.15.7562.PubMed CentralView ArticlePubMedGoogle Scholar
- Font J, Mackay JP: Beyond DNA: zinc finger domains as RNA-binding modules. Methods Mol Biol. 2010, 649: 479-491. 10.1007/978-1-60761-753-2_29.View ArticlePubMedGoogle Scholar
- Mittal N, Roy N, Babu MM, Janga SC: Dissecting the expression dynamics of RNA-binding proteins in posttranscriptional regulatory networks. Proc Natl Acad Sci U S A. 2009, 106 (48): 20300-20305. 10.1073/pnas.0906940106.PubMed CentralView ArticlePubMedGoogle Scholar
- Chenn A, Walsh CA: Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science. 2002, 297 (5580): 365-369. 10.1126/science.1074192.View ArticlePubMedGoogle Scholar
- Grigoryan T, Wend P, Klaus A, Birchmeier W: Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mutations of beta-catenin in mice. Genes Dev. 2008, 22 (17): 2308-2341. 10.1101/gad.1686208.PubMed CentralView ArticlePubMedGoogle Scholar
- Zechner D, Fujita Y, Hulsken J, Muller T, Walther I, Taketo MM, Crenshaw EB, Birchmeier W: Birchmeier C: beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev Biol. 2003, 258 (2): 406-418. 10.1016/S0012-1606(03)00123-4.View ArticlePubMedGoogle Scholar
- Greenbaum D, Colangelo C, Williams K, Gerstein M: Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol. 2003, 4 (9): 117-10.1186/gb-2003-4-9-117.PubMed CentralView ArticlePubMedGoogle Scholar
- Nie L, Wu G, Zhang W: Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: a quantitative analysis. Genetics. 2006, 174 (4): 2229-2243. 10.1534/genetics.106.065862.PubMed CentralView ArticlePubMedGoogle Scholar
- Shin S, Mitalipova M, Noggle S, Tibbitts D, Venable A, Rao R, Stice SL: Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem Cells. 2006, 24 (1): 125-138. 10.1634/stemcells.2004-0150.View ArticlePubMedGoogle Scholar
- Kiesslich A, von Mikecz A, Hemmerich P: Cell cycle-dependent association of PML bodies with sites of active transcription in nuclei of mammalian cells. J Struct Biol. 2002, 140 (1–3): 167-179.View ArticlePubMedGoogle Scholar
- Keene JD, Tenenbaum SA: Eukaryotic mRNPs May represent posttranscriptional operons. Mol Cell. 2002, 9 (6): 1161-1167. 10.1016/S1097-2765(02)00559-2.View ArticlePubMedGoogle Scholar
- Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G: GO::TermFinder—open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics. 2004, 20 (18): 3710-3715. 10.1093/bioinformatics/bth456.PubMed CentralView ArticlePubMedGoogle Scholar
- Hines WC, Bazarov AV, Mukhopadhyay R, Yaswen P: BORIS (CTCFL) is not expressed in most human breast cell lines and high grade breast carcinomas. PLoS One. 2010, 5 (3): e9738-10.1371/journal.pone.0009738.PubMed CentralView ArticlePubMedGoogle Scholar
- Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000, 25 (2): 169-193. 10.1677/jme.0.0250169.View ArticlePubMedGoogle Scholar
- Camacho-Vanegas O, Weighardt F, Ghigna C, Amaldi F, Riva S, Biamonti G: Growth-dependent and growth-independent translation of messengers for heterogeneous nuclear ribonucleoproteins. Nucleic Acids Res. 1997, 25 (19): 3950-3954. 10.1093/nar/25.19.3950.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.