1 Supplementary information
Material and Methods Plasmid construction
For the construction of the Egr1 expression plasmid, the pCMV-SPORT6-Egr1 vector (Invitrogen), containing full length human Egr1 cDNA, was digested with KpnI and NotI and inserted between the corresponding restriction sites of pcDNA3.1 (Invitrogen) to generate pcDNA3.1-Egr1. For the construction of short hairpin RNAs (shRNA) that target Egr1, shEgr1-sense and shEgr1-antisense oligonucleotides (Supplementary Table S1A) were annealed and inserted between the BbsI/EcoRI sites of pmU6 to generate pmU6(mouse U6)-shEgr1. This product was digested with BamHI and EcoRI and inserted between these sites in pLSP to generate the lentiviral vector, pLSP-shEgr1. For the construction of the pmU6-miR-199a-5p/-3p expression plasmid, a DNA fragment corresponding to the region from 46 bp upstream to 48 bp downstream of the hsa-miR-199a-2 stem loop structure defined in miRBase was PCR amplified from H-III-TC genomic DNA using the primers 199a-2 f and 199a-2 r (Supplementary Table S1B). The PCR product was then cloned into pCR2.1
(Invitrogen) to generate pCR2.1-miR199a-5p/-3p. This was then digested with BbsI and EcoRI and subcloned into pmU6 to generate pmU6-miR-199a-5p/-3p. This was further digested with BamHI and EcoRI and subcloned into pLSP to generate the lentiviral vector, pLSP-miR-199a-5p/-3p.
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previously described protocol (21). The synthesized deoxynucleotide pair, TuD5p-s and TuD5p-as (or TuD3p-s and TuD3p-as) are listed in Supplementary Table S1C. For the construction of the miPPR-199a-2 luciferase reporter plasmids, full length miPPR-199a-2 DNA was amplified from TIG-112 genomic DNA using the primer pair, mir199a-2 f1 and r1 (Supplementary Table S1D). The resulting PCR product was then inserted into pCR2.1 to generate pCR-miPPR-199a-2-WT, which was further digested with EcoRV and EcoNI. The fragment ends were blunted with Klenow fragment and inserted into the EcoRV site of pGL4.12 (Promega, Madison, WI) to generate
pGL-miPPR-199a-2-WT. Using pGL-miPPR-199a-2-WT, a series of substitution mutants within the putative Egr1 binding sites were generated via site-directed mutagenesis using the primer pairs m1-f and -r, m2-f and -r, and m3-f and –r to yield pGL-miPPR-199a-2-m1, -m2 and -m3 (Supplementary Table S1D), respectively. These fragments were cloned into pCR2.1 to generate
pCR-miPPR-199a-2-m1, -m2, and -m3 which were then digested with EcoRV and HindIII and inserted into pGL4.12 to generate pGL-miPPR-199a-2-m1, -m2, and -m3, respectively. After substituting the 2.7 kb XbaI fragment of pGL-miPPR-199a-2-m2 with that of
pGL-miPPR-199a-2-m1, the 0.5 kb ApaI fragment of the resulting plasmid was substituted with that of pGL-miPPR-199a-2-m3 to generate pGL-miPPR-199a-2-m4, in which all three putative Egr1 binding sites had been substituted.
For the construction of psi-CHECK2 (Promega) luciferase reporter plasmids, deoxynucleotide pairs containing the complementary sequences of miR-199a-5p or miR-199a-3p were synthesized
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(Supplementary Table S1E). After annealing these pairs, the fragments were inserted between the XhoI/PmeI sites of psi-CHECK-2. As negative controls, substitution mutants were also constructed using the same procedure (Supplementary Table S1E). For the construction of Brm 3’ UTR luciferase reporter plasmids, the full length 3’ UTR of Brm was PCR-amplified from TIG-112 genomic DNA using the primer pair Brm 3’UTR f1 and r1, (Supplementary Table S1F), and was inserted into pCR2.1 to generate pCR-Brm 3’UTR-WT. The BamHI/XbaI fragment of pCR-Brm 3’UTR-WT was then inserted into pTK4.12(c.P-) to generate pTK-Brm 3’UTR-WT. Corresponding mutants were generated from pCR-Brm 3’UTR-WT by overlapping PCR using the primers listed in Supplementary Table S1F to generate pTK-Brm 3’UTR-m1, m2 and m1/m2. For the construction of the
pGL4.12-Egr1-pro-CMV-blasticidin reporter plasmid, a DNA fragment covering the region between -1293 and +153 around the Egr1 gene was PCR amplified from C33A genomic DNA using the primer pair pro-for and pro-rev (Supplementary Table S1G). The PCR product was cloned into PCR2.1 to generate pCR2.1-Egr1 pro. This was digested with KpnI and XhoI and inserted into pGL4.12 which was further digested with PciI and BglⅡand inserted into the PciI/BglⅡsite of pGL4.12-CMV-blastcidin to generate pGL4.12-Egr1 pro-Luc-CMV-blasticidin. This was further digested with StuⅠ(or BglⅡ), the fragment of which was blunt-ended and digested with BglⅡ. This fragment was inserted into Xho (blunt-ended)/BglⅡto generate pGL4, 12-Egr1 pro
4 Western blotting
Total protein extracts were prepared by boiling the cells in SDS sample gel buffer for 10 min at 95°C. The proteins were then separated by 5-20% SDS-PAGE and transferred onto Immobilon-P PVDF membranes (Millipore). Immunoblotting was performed by incubating the membrane overnight at 4°C with primary antibodies: anti-Brm (ab15597; Abcam), anti-BRG1 (H88; Santa Cruz
Biotechnology, Santa Cruz, CA), anti-Egr1 (sc-189; Santa Cruz Technology), anti-IKKβ (#2684; Cell Signaling), anti-versican (sc-25831; Santa Cruz Biotechnology), anti-fibronectin (610078; BD Transduction Laboratories), and anti-β-actin (610538, BD Biosciences). After three washes with phosphate-buffered saline containing Tween20, secondary antibodies (anti-Rabbit Donkey antibody HRP, AP182P; and anti-Mouse Donkey antibody HRP, AP192P; Chemicon) were incubated with the membranes for 1 h at room temperature. Signals were detected using ECL reagent (Amersham) or ImmunoStar LD (Wako).
Quantification of RT-PCR
For quantitative analysis, PCR bands were quantified using a Luminescent Image Analyzer (LAS4000, Fujifilm) after polyacrylamide gel electrophoresis and appropriate PCR cycle numbers were determined so that the calibration curve using samples that gave the highest values should be almost linear. The expression of miR-199a-5p/-3p and miR-214 was determined by qRT-PCR (7300 Real Time PCR system, Applied Biosystems) using miRNA-specific looped RT-primers and the
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TaqMan probes as recommended by the manufacture (Applied Biosystems). U6 snRNA was used as an internal control.
Chromatin immunoprecipitation
ChIP analysis was performed using a commercial assay kit (Upstate Biotechnology) in accordance with the manufacturer’s instructions using SW13 and MDA-MB435 cells (1×107).
Immunoprecipitations were performed overnight at 4°C with 10 µg of anti-Brm, anti-BRG1, or anti-Egr1 antibodies, or non-immunized rabbit IgG as a control. Following digestion with Proteinase K, DNA was phenol-extracted from the immunoprecipitates and precipitated with isopropanol. Using this prepared DNA in each case as a template, semi-quantitative PCR analysis was performed using the primer pairs listed in Supplementary Table S2B.
Primer extension and dideoxy sequencing
Total RNA from SW13 and MDA-MB435 cells were isolated using Isogen (Nippon Gene) and treated with DNase. PolyA RNA was extracted from these samples using Poly(A) Purist (Ambion). All procedures were performed as described previously (31).
In situ hybridization
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sections was performed as described previously (36). Use of the clinical tissue sections in this study was approved by the Fujita Health University ethical review board for human investigation.
Immunohistochemistry
Deparaffinization, endogenous peroxidase inactivation, antigen retrieval from formalin fixed paraffin embedded clinical tissues, and immunostaining of Brm was performed as described previously (14). For Egr1 immunostaining, the sections were incubated overnight at room temperature with
anti-EGR1 antibody (1:1000, #4153 Cell Signaling Technology) and washed in PBS. Anti-FITC horseradish peroxidase–conjugated antibodies (DAKO,P5100) at a 1:100 dilution in TBS/1% bovine serum albumin were then applied to the slides for 30 min at room temperature, followed by three washes in TBS/0.1% Tween 20 (TBS-T). For signal amplification, FITC-conjugated phenol (fluorescyl-tyramide; DAKO, K1497) was applied to the slides for 15 min at room temperature, followed by three washes in TBS-T. Finally, an anti-FITC antibody conjugated to horseradish peroxidase (DAKO,K1497) was added to the slides for 15 min at room temperature, followed by three washes in TBS-T. The reaction products were visualized using a 50 mg/dL
7 Supplementary Figure legends
Supplementary Fig. S1.
Structure of the chemically modified miRNA duplexes used for strand specific loading onto RISCs. The 5’-terminus of one strand in each miRNA duplex was modified with
1-(3,5-bishydroxymethylphenyl)-4-(dimethylamino)naphthalene (DANap). The strand pairs were annealed at 95°C to create miRNA duplexes.
Supplementary Fig. S2. Structure of the miR-199a-5p/-3p expression vector driven by mouse U6 (mU6) promoter and its functional analysis by reporter assay. A. Schematic representation of pmU6-miR-199a-5p/-3p, an expression plasmid vector for RNA containing the entire
pre-miR-199a-2 product. The sequences of miR-199a-5p and miR-199a-3p are indicated in red and blue, respectively. B. The expression of mature miR-199a-5p and -3p from pmU6-miR-199a-5p/-3p was confirmed by luciferase reporters (upper panel) containing sequences that are fully
complementary to miR-199a-5p or -3p in the Luc 3’ UTR region as well as their mutant versions in the seed sequences. PmU6-miR-199a-5p/-3p (100ng) was transfected into HeLaS3 cells in 24-well plates with reporter plasmids (100ng each). Luciferase activities were measured at 48 h
post-transfection. PmU6-sh-lacZ was used as a control vector. Data are the means ±SD from triplicate experiments (lower panel). 5p reporter activity, but not 5m activity, was strongly and specifically repressed by both pmU6-miR-199a-5p/-3p. Likewise, 3p but not 3m reporter activity
8 was repressed by pmU6-miR-199a-5p/-3p.
Supplementary Fig. S3.
Structure and function of expression plasmid vectors for TuD-miR199a-5p and TuD-miR199a-3p. A. RNA sequences of TuD-miR199a-5p and TuD-miR199a-3p. The binding regions for the seed
sequences of miR-199a-5p and miR-199a-3p are marked in red and blue, respectively. The four nucleotides in the box will form a bulge when TuD-RNA hybridizes to the corresponding miRNA. The bulge has been shown to prevent efficient cleavage of TuD RNA by Ago proteins. B. Selective knockdown of miR-199a-5p and -3p by TuD-miR199a-5p andTuD-miR199a-3p, respectively. Using the luciferase reporters shown in Supplementary Fig. S1, the suppressive effects by TuD RNAs were evaluated. psL1180-U6-TuD vectors (100ng each) were transfected into HeLaS3 cells in 96-well plates together with the reporter plasmids (200ng) and pmU6-miR-199a-5p/-3p vector (200ng). Luciferase activities were measured at 48 h post-transfection. psL1180-U6-NC (Negative Control) was used as a negative control. Data are the means ±SD from triplicate experiments. In control transfectants (TuD-NC), luciferase activity was significantly repressed from either the 5p or 3p reporter, but not significantly affected from the 5m or 3m reporters. The introduction of
TuD-miR199a-5p rescued the luciferase-activity suppression caused by miR-199a-5p for the 5p reporter but not the 3p reporter, indicating that TuD-miR199a-5p selectively knocks down miR-199a-5p. Likewise, for the 3p reporter, TuD-miR199a-3p specifically recovers the reduction
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Supplementary Fig. S4.
Primer extension analysis to determine the transcriptional start site (TSS) of the miR-199a-2 gene. On the right, the products of dideoxynucleotide sequencing of the antisense strand using the same primers were co-migrated to determine the precise TSS. On the left, the sequence of the template strand is shown. The arrowheads indicate the major TSSs in SW13 cells, which express mature miR199a-5p and -3p.
Supplementary Fig. S5.
Sequence around the miPPR-199a-2 region. Sequence highlighted in gray is that of miPPR-199a-2. The arrows within miPPR-199a-2 indicate the two transcriptional start sites (TSS) detected by primer extension analysis. One of TSS, the C residue was designated as +1. Primers a-c were used in the RT-PCR (primer a, b and c; lower) and primer extension assays (primer c; Supplementary Fig. S4). In RT-PCR analysis of the primary transcripts of the miR-199a-2 gene, the b-c primer pair amplifies a product when total RNA from SW13 cells is used as the template. Right panel, the same primer pairs was used for genomic PCR as positive controls.
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binding sites of several transcriptional factors as detected by TRANSFAC Alibaba 2.1. There are three putative Egr1 binding sites (#1, #2 and #3) in miPPR-199a-2.
Supplementary Fig. S7. Exogenous Egr1 induces the expression of endogenous miR-199a-5p and positively regulate miR-199a-2 reporter gene expression.
(A) MDA-MB435 cells were transfected with an empty vector or an Egr1 expression vector. Expression levels of Egr1 and β-actin protein (left) and of mature miR-199a-5p RNA (right) were analyzed as described in the legend of Figure 3A. (B) Dosage dependency of miR-199a-2 reporter gene activity upon Egr1. MDA-MB435 cells were co-transfected with pGL4.12-miPPR-199a-2 (250ng), pGL4.74 (5ng) and increasing amounts of an expression vector for Egr1 along with the appropriate amounts of empty vector to control for the transfected DNA content (755ng). After 48 hours, the dual luciferase activity was determined. Data are the means ±SD from triplicate
experiments.
Supplementary Fig. S8
ChIP analysis of the human miPPR-199a-2 region in AZ521. DNA isolated by chromatin
immunoprecipitation with Egr1 antibodies (α-Egr1) and non-immunized rabbit IgG was used. PCR was performed using primer pairs specific for the #1, #2 or #3 Egr1 binding sites as well as for intron 2 of C2TA and IFITM1 promoter.
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Supplementary Fig. S9. A double-negative feedback loop is formed between miR-199a and Brm. Expression patterns of the mature miRNAs, miR-199a-5p, -3p and miR-214, miR199a-2/214 transcripts, Egr1, Brm, BRG1 mRNAs, and the Brm and BRG1 proteins in the human cancer cell lines were analyzed. The relative expressions of miR-199a-5p, -3p and miR-214 are normalized to that of 293FT cells. SW13 was used as the internal positive control as in Figure 5A.
Supplementary Fig. S10 Expression levels of Egr1, Brm and BGR1 proteins in a serious of human cancer cell lines. Protein gel bands detected by western blotting were quantified by Las4000 and (bar graphs). After normalization using the actin band intensity, the averages (n=2-3) of the relative Egr1 expression levels to SW13 and the relative Brm (or BRG1) levels to KB were calculated using several gels including those shown in Figures 3 and S9.
Supplementary Fig. S11 Egr1 promoter analysis for the detection of regions responsible for its suppression by Brm. Two truncated versions of pGL. 4.12-Egr1 promoter-Luc-CMV-B1a were constructed and stably introduced into MDA-MB435 cells as described in the legend of Figure 4C. The putative binding sites for C/EBPα, C/EBPβ, NF-IB and NRSF are shown (upper panel). shBrm, shBRG1 and shGFP vectors were transiently transfected into each stable transformant as described for Figure 4C. Data are the mean±SD of three independent experiments. *P<0.05.
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We have previously reported in human non-small cell lung carcinoma cell lines that a protein complex including neuron-restrictive silencer factor (NRSF) binds SWI/SNF complex and recruits the resulting larger complex to neuron specific gene promoters and suppresses them (10). It is also known that such regulators as C/EBPα, C/EBPβ and NF-IB, which can associated with the
Brm-SWI/SNF complex, often function as negative regulators. Importantly, an Egr1 promoter region responsive to suppression by Brm (-1293～-502) contains putative binding sites for these negative regulators.
Supplementary Fig. S12. In situ hybridization analysis using FITC-labeled LNA-modified probes to detect miR-199a-5p or -3p expression in formalin-fixed human cancer cell lines. Cytoplasmic miR-199a-5p or -3p are highlighted by the detectable FITC signal (green). Nuclear DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue).
Supplementary Fig. S13.
HDAC inhibitor treatments alter the expressions of miR-199a-5p/-3p, Egr1 and Brm. SW13 cells were treated with the HDAC inhibitors FK228 (0.7ng/ml; a gift from Fujisawa Corporation, Osaka, Japan) or CHAP31 (12nM; a gift from the Japan Energy Corporation, Saitama, Japan). After 72 h, total protein and total RNA extracts were prepared. MiR-199a-5p, -3p and Egr1 expressions were analyzed by RT-PCR. Brm and β-actin proteins were analyzed by western blotting.
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We have previously reported that all cancer cell lines deficient in Brm can recover its expression for two weeks following transient treatments with HDAC inhibitors such as FK228 or CHAP31, whereas Brm suppression occurs at the post-transcriptional level. Interestingly, in our present experiments these treatments caused a reduction in the levels of mature miR-199a -5p/-3p and Egr1 mRNA, which are associated with Brm induction as observed in our previous studies. These results still do not indicate the precise targets of the HDAC inhibitors, but confirm that they drastically alter the cells from an miR-199a dominant state to a Brm dominant state within the double-negative feedback regulatory network.
