Conceived and designed the experiments: TT ZZ WW. Performed the experiments: LX JW MW YS. Analyzed the data: MW. Wrote the paper: LX JW MW WW.
The authors have declared that no competing interests exist.
Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for
All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of
Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence
It is well known that p16INK4a inhibits cdk (cyclin-dependent kinase)4/cdk6-mediated phosphorylation of retinoblastoma gene product (pRb) and induces cell cycle arrest in G1 phase
Human embryonic lung fibroblast cell line (2BS cells) obtained from the National Institute of Biological Products (Beijing, China), was cultured in DMEM medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 1 µg/ml streptomycin
To make pGL3-620, 620bp 5′-fragment of human
GC-box/probe | Position | Sequence |
GC-I | −474–−447 | Wild type: 5′-GGAAGGAAACGGGGCGGGGGCGGATTTC-3′ |
Mut 1: 5′-GGAAGGAAAC |
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GC- II | −462–−435 | Wild type: 5′-GGCGGGGGCGGATTTCTTTTTAACAGAG-3′ |
Mut 2: 5′-GGCGGGGGC |
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GC-III | −380–−355 | Wild type: 5′-GGGAGGCCGGAGGGCGGTGTGGGGGG-3′ |
Mut 3: 5′-GGGAGGCC |
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GC-IV | −76–−49 | Wild type: 5′-CAGAGGGTGGGGCGGACCGAGTGCGCTC-3′ |
Mut 4: 5′-CAGAGGGTGGGG |
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GC-V | −26–−1 | Wild type: 5′-GCAGGCAGCGGGCGGCGGGGAGCAGC-3′ |
Mut 5: 5′-GCAGGCAGCG |
All plasmids were purified with Qiagen Plasmid Midi Kits (Chatsworth, CA). For each transfection experiment, 2BS cells were seeded at 12-well plates and grown for about 24 h until they were above 90% confluent, then transfected with an equal amount of reporter plasmid (1.6 µg) and 0.32 µg of pRL-CMV (Promega) as transfection efficiency control, using lipofectamine 2000 (Invitrogen) and following the manufacturer's indications. Five hours later, serum-free DNA-containing medium was replaced by fresh growth medium and the cells were harvested 48h after transfection. Luciferase assays were performed as described (Dual-Luciferase Reporter Assay System, Promega). All assays were carried out in triplicate and performed twice for confirmation.
Total RNA was prepared from exponentially growing cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). For Northern blot analysis, RNA was electrophoresed in 1.5% formaldehyde-denaturing agarose gel, with the 0.24–9.5 kb RNA Ladder included in one lane as a size marker (Gibco BRL, Gaithersburg, MD). RNA was then transferred onto Biodyne B membrane (Pall, East Hills, NY) according to manufacturer's recommendations and fixed. The human p16INK4a gene-coding region was used as probe labeled with [α-32P] dCTP (Yahui, Beijing, China) by random priming using the Prime-a-Gene Labeling System (Promega). Hybridization was carried out in ExpressHyb™ Hybridization Solution (Clontech) at 68°C for 1 h. The blot was stringently washed with 0.1×SSC, 0.1% SDS at 68°C for 15 min twice. Autoradiography was performed at −80°C. The same blot was stripped after probing and then reprobed with the 32P-labeled GAPDH probe to control for equivalent RNA loading in each lane.
Cell extracts were prepared following standard procedures. Briefly, three to five volumes of lysis buffer (50 mM Tris-HCL, pH 7.4, 0.25 M NaCl, 0.1% Triton-X-100, 1 mM EDTA, 50 mM NaF, 1 mM DTT, 0.1 mM Na3VO4) were added to a cell pellet. The following protease inhibitors were added: 0.1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin). After incubation on ice for 30 min, samples were centrifuged at 14,000 r.p.m. for 5 min at 4°C to recover the supernatant. After proteins were electrophoresed in a 15% (for p16) or 8% (for Sp1/Sp3) denaturing polyacylamide gel and transferred to a PVDF membrane, the membrane was blocked in 5% nonfat milk-TBS-0.25% Tween 20 for 1 h and incubated with the primary antibody in TBS-0.25% Tween 20 for 1 h at room temperature. Complexes were detected with horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence (SuperSignal, Pierce). The primary and secondary antibodies used in this study were all from Santa Cruz Biotechnology (Santa Cruz).
Nuclear extracts from 2BS cells were prepared as follows: About 1×106 cells were harvested, washed twice with cold PBS (phosphate-buffered saline) and collected by centrifugation at 3500 rpm at 4°C for 5 min. The cells were resuspended in 400 µl of solution I (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF) and lysed by passing them through a 25 gauge syringe. Nuclei were pelleted at 2,000 r.p.m for 10 min, washed once with solution I and resuspended in 200 µl of solution II (solution I with 5% glycerol, 400 mM NaCl and without KCl). The suspension was rotated at 4°C for 30 min and then centrifuged at 14,000 r.p.m, 4°C, for 30 min. The resulting clear supernatant was stored at −80°C until use.
A 38 bp long oligonucleotide 5′-GGAAGGAAACGGGGCGGGGGCGGATTTCTTTTTAACAG-3′ (oligo I) extending from nt −474 to −437 or a 28 bp long oligonucleotide 5′-CAGAGGGTGGGGCGGACCGAGT GCGCTC-3′ (oligo II) extending from nt −76 to −49 on the 5′ UTR of p16 gene, was used in EMSA and supershift assay. Double stranded DNA fragments were end-labeled with [γ-32P] dATP (Yahui, Beijing, China) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The probes were purified using QIAquick Nucleotide Removal Kit (Qiagen) and 15,000 cpm were incubated for 25 min on ice with 10 µg of nuclear extract from 2BS cell line, in the presence of 20 mM Tris-HCl pH 7.5, 75 mM KCl, 3.5 mM DTT, 20 nM ZnCl2, 1 µg/µl BSA, 5% glycerol and 1 µg poly (dI-dC) (Pharmacia Biotech, Piscataway, NJ) in a total volume of 20 µl. For supershift experiments, 2 µg of antibody against Sp1 or Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 20 min on ice with the nuclear extract before adding the labeled probe. DNA/protein complexes were separated from free DNA on a 5% polyacrylamide gel in TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 4°C for 120 min at 340 V. After electrophoresis, gels were dried and autoradiographed.
ChIPs were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate, New York) according to manufacturer's instruction. In brief, 1×106 cells were crosslinked by adding formaldehyde directly to cell culture media and incubated for 10 min at 37°C. Wash cells twice with cold PBS and then cells were scraped and resuspended in 200 µl of SDS Lysis Buffer. Chromatin was then sonicated to an average length of 0.5 Kb for three 30 sec. pulses at maximum power. Chromatin extracts were diluted 10 folds in Dilution Buffer and preincubated for 30 min at 4°C with 80 µl of Salmon Sperm DNA/ protein A Agarose. Twenty microlitres of diluted supernatant was kept for isolation of input DNA and to quantitate the DNA in different samples. After pelleting agarose by brief centrifugation, 2 µg of anti-Sp1 antiserum (test group) or 2 µg of β-actin antibody (irrelevant antibody control) was added to the supernatant fraction and incubated overnight at 4°C with rotation. In addition, perform a no antibody immunoprecipitation by incubating the supernatant fraction with Salmon Sperm DNA/ protein A Agarose for 1 h at 4°C. Add 60 µl of Salmon Sperm DNA/ protein A Agarose for 1 h at 4°C to collect the antibody/antigen-DNA complex. The chromatin bound to the protein A Agarose beads was eluted in 500 µl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). After reversing crosslinks, the samples were deproteinized and phenol-chloroform extracted, then DNA was ethanol precipitated using yeast tRNA as a carrier. Pellets were resuspended in 50 µl of TE buffer for PCR analysis.
Each PCR reaction mixture contained 5 µl of immunoprecipitated chromatin in a final reaction volume of 20 µl. PCR mixtures were amplified for 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s. To amplify GC-box containing regions of
The target sequence against mRNA of Sp1 is 5′-AUCACUCCAUGGAUGAAAUGATT-3′, which has been reported to be effective in some cell lines
Cells were transfected using Lipofectamin 2000 as specified by the manufacturer. The transfection mixture was left on the cells for 4 h, after which DMEM/20% serum without antibiotics was added. For efficient knockdown two more transfections were performed at 24 h and 48 h after the first transfection.
Cells were washed twice in PBS, fixed in 3% formaldehyde, and washed again in PBS. The cells were incubated overnight at 37°C (without CO2) with freshly prepared SA-β-Gal staining solution.
To determine the crucial GC-rich region of the human
A. Schematic presentation of mutants of GC-boxes in the
To evaluate whether Sp proteins indeed binds to the GC boxes, gel electrophoretic mobility shift assays (EMSA) were performed. Oligo I including GC-I and GC-II and oligo II containing GC-IV were 5′end labeled with [γ-32P-]dATP and incubated with nuclear extracts from either young or senescent 2BS cells. The DNA-protein binding complexes were then analyzed on 5% polyacrylamide gel. As shown in
Electrophoretic mobility shift assays (EMSA) was performed using nuclear extracts (NE) from young (Y) or senescent (S) 2BS cells and radiolabeled oligo I (A) or oligo II (B). Competition was performed in the presence of 100-fold molar excess of the cold synthetic oligos (Comp) as indicated. The major specific complexes are indicated as a, b and c. The presence of Sp1 and Sp3 in the DNA-protein complexes was monitored by the disappearance of the retarded bands in the presence of antibodies against Sp1 and Sp3 (supershift).
We next want to know whether Sp1 and/or Sp3 could bind to
A and B. ChIPs assays of young (Y: PD27) and senescent (S: PD60) 2BS cells using antibodies against Sp1 (A) or Sp3 (B), antibody against β-actin was used as irrelevant control (Nc). (C) Senescent (S: PD60) 2BS cells were treated with MTR in the dosage indicated, 24 hours later, cells were harvested and subjected to ChIP assays. Data are representative of three independent experiments.
To analyze the effect of Sp1 and Sp3 on
The reporter construct pGL3-620 was co-transfected with pCMV-Sp1 or/and pCMV-Sp3 or control vector along with pRL-CMV in young (A) and senescent (B) 2BS cells. Luciferase assays were performed and normalized to the Renilla luciferase activity. The mean±S.E. from three independent experiments was used to express the relative luciferase activity.
To confirm the effect of Sp1 on
Young (A) and senescent (B) 2BS cells were transfected with pGL3-620. 24 hours after transfection, cells were exposed to different dosage of MTR (M-A) as indicated for 24 more hours and subjected to luciferase activity assays. The data represent the means±S.E. of five independent experiments.
Young (Y) and senescent (S) 2BS cells were either treated with 100 nM MTR (MTR) for 24 hr or left untreated, total RNA and protein were prepared and subjected to analyze the expression of indicated genes by Northern blotting (A) and Western blotting (B), respectively.
As the mechanism of MTR treatment is blocking the transcription factors such as Sp family binding to GC-box, in this way, MTR could also affect the binding of other transcriptional factors to
Following transfection of 2BS cells with si-Sp1 or a control plasmid, RT-PCR (A) and Western blotting (B) were carried out to analyze the expression of the genes indicated.
The results from two aspects mentioned above suggest that not only Sp1 can activate
The results mentioned above demonstrate that during the cell aging process, Sp1 contributes to the higher expression of
Western blot analysis of Sp1 and Sp3 expression in young (Y) and senescent (S) 2BS cells, data are representative of three independent experiments.
SA-β–galactosidase staining is a common marker for cellular senescence. Usually, the β-galactosidase activity increases with the cell PD(population doubling) accumulating. The biological effect of over-expression (by Sp1 expression plasmid transfection) or knockdown (by RNAi approach) of Sp1 in 2BS cells were further evaluated by this method. The results showed that Sp1-overexpressed cells were strongly stained blue versus the control. However, there were only a few dispersed cells that were SA-β-Gal-stained in the Sp1 knocked-down cells (
2BS/pCMV (A), 2BS/ pCMV-Sp1 (B), 2BS/pSliencer (C), 2BS/si-Sp1 (D), (all above at PD 40), untransfected young (PD 27) (E), middle-aged (PD 48) (F), and senescent (PD 56) (G) 2BS cells were cultured and then stained to assess SA-β-Gal activity.
Protein binding studies identified Sp1 and Sp3 as the major components of the complexes formed between the nuclear extracts and the oligos containing GC box. Although we cannot preclude involvement of other factors, the Sp1 and Sp3 antibodies blocked each of the specific complexes in vivo and in vitro. Sp1 is a widely studied transcription factor that can bind to and act through the GC boxes. Although it is generally believed that Sp1 is ubiquitously expressed, Sp1 gene expression can show up to 100-fold differences in different cell types and at different stages of development in mouse
It has been documented that the expression of
In addition, though Sp1 is considered to be a constitutive transcription factor, Sp1 protein levels can vary significantly in different tissues. To test whether increased Sp1 binding is a result of induced Sp1 expression in senescent cells, we performed Western analyses. The results showed that the protein levels of Sp1 and Sp3 did not change significantly between young and senescent 2BS cells. So the increased binding observed in the senescent cells is likely the result of the augmentation of Sp1 and/or Sp3 binding affinity. On the other hand, the result of RNAi transfection demonstrated that although it need not increase Sp1 expression, however, a basal level is necessary for
The alteration of Sp1 activity during senescence may result from different post-translational modification or transcriptional co-factor. The two major types of modification that are thought to be involved in transcription regulation by Sp1 are glycosylation and phosphorylation. O-glycosylation of Sp1 with N-acetylglucosamine has been linked to multiple changes in Sp1 function, including altered self-association, altered interaction with basal transcription factors and modulation of its degradation
All in all, among the multitudinous transcription factors, Sp1 is also an important and essential member for the transcription and expression of
We thank Dr Robert Tjian, Dr Guntram Suske and Dr Yongfeng Shang for providing the vectors. We also thank B-Match Consulting for manuscript revision.