Effect of SMAR1 overexpression on G1/S transition molecules.
Tumor suppressor proteins have been shown to directly alter cell cycle regulation (48
). Earlier we reported that SMAR1 regresses the B16-F1-induced tumor in the mouse model (18
). To investigate the role of SMAR1 in regulating the cell cycle, we analyzed the expression levels of different cyclins. Flag-SMAR1 was overexpressed in asynchronous culture of B16-F1 cells and harvested at 24 h. Protein levels of cyclin D1 and cyclin E were downregulated in SMAR1-overexpressed compared to mock-transfected cells. There was no change observed in cyclins A, B1, and D3 (Fig. ). A human homolog of SMAR1, BANP, is shown to be located at 16q24, the locus that has gained importance as it harbors at least three breast tumor suppressor proteins. Since SMAR1 downregulated cyclin D1, whose amplification is seen in many breast cancers, we were interested to study the role of SMAR1 in human breast cancer cell lines. Thus, SMAR1 was overexpressed in non-breast cancer (293) and breast cancer (MCF-7, HBL-100, and MDA-MB-468) cell lines. Upon overexpression of SMAR1 in 293 cells, cyclin D1 and D3 downregulation started at 16 h and continued until 36 h. No change in the levels of cyclin B1 was observed. The effect of the decrease of cyclin D1 levels on the cyclin D1/CDK4 complex was verified in the same lysates by checking the phosphorylation status of Rb. As expected, a decrease in the phosphorylation of Rb (pRb s807/811) was observed from 16 to 36 h; however, the total Rb remained unaltered. A reverse pattern of p27/kip1
expression was seen in the case of SMAR1 overexpression. STAT5 transcription factor, under phosphorylated conditions, gets recruited to the cyclin D1 promoter and favors transcription (27
). SMAR1 overexpression reduced the phosphorylation of Stat-5 drastically. Considerable downregulation of CDK4 was also noticed in the case of SMAR1 overexpression (Fig. ). Overexpression of Flag-SMAR1 in 293 cells was confirmed using Flag antibody (Fig. ).
FIG. 1. SMAR1 inhibits cyclin D1 expression. (A) Expression of various cyclins was checked on mock (lane 1) and Flag-SMAR1 (lane 2) transfections in B16-F1 cells. (B and C) Western blot analysis for time course overexpression of Flag-SMAR1. Expression patterns (more ...)
In a similar experiment of Flag-SMAR1 overexpression in MCF-7 cells, a fivefold downregulation of cyclin D1 was observed at 16 h, and there was no detectable protein at 24 h (Fig. ). As indicated, cyclin D3 was also downregulated in MCF-7, but not drastically. Overexpression of SMAR1 was verified in MCF-7 cells by anti-Flag immunoblotting (Fig. ). Similar results of cyclin D1 downregulation were seen in HBL-100 cells along with a strong repression of cyclin D3 (Fig. ). In the case of MDA-MB-468, cyclin D1 but not cyclin D3 was downregulated (Fig. ). Thus, SMAR1 overexpression downregulated cyclin D1 irrespective of cell type. To further verify the effect of SMAR1, stable clones expressing Flag-SMAR1 or Flag vector in MCF-7 cells were checked for expression of cyclin D1. Transfected cells were stably selected using neomycin. Cells stably expressing SMAR1 showed downregulation of cyclin D1, but no change in cyclins D2 and D3 was observed (Fig. ). Thus, ectopic expression of SMAR1 downregulates cyclin D1 expression.
Expression profile of SMAR1 in various breast cancer and non-breast cancer cell lines.
Earlier studies using cancer cell lines have reported that the expression of SMAR1 is defective in the majority of cancer cell lines (18
). As cyclin D1 expression is dysregulated in a majority of the breast tumors and breast cancer cell lines, we wanted to assess if the upregulation of cyclin D1 is accompanied by downregulation of SMAR1 in breast cancer cell lines.
Transcript analysis revealed that SMAR1 was reduced in MCF-7, HBL-100, MDA-MB-231/468, SK-BR3, and T-47D compared to 293. There was no detectable transcript in ZR75.1 and ZR75.30 (Fig. ). Further validation was done using real-time RT-PCR analysis. The graph shown in Fig. was obtained by repeating the experiment thrice with different sets of cDNA samples. SMAR1 was downregulated by 12-fold in the MCF-7 cell line, while 18-fold downregulation was seen in HBL-100; 10- to 7-fold variations in MDA MB-231/468 and 3-fold differences in T-47D cells compared to 293 and B16-F1 were also seen. No amplification was seen in ZR75.1 and ZR75.30. Immunoblotting using SMAR1 antibody showed lowered protein expression in T-47D and SK-BR-3, while protein expression was undetectable in MCF-7, ZR75.1, ZR75.3, HBL-100, MDA-MB-231, and MDA-MB-468. SMAR1 expressing T-47D and SK-BR3 showed low cyclin D1 levels, while a high induction of cyclin D1 was seen in MCF-7, ZR75.1, ZR75.3, HBL-100, and MDA-MB-231/468 that showed drastically reduced levels of SMAR1 (Fig. ).
FIG. 2. Expression of SMAR1 and cyclin D1 in various breast cancer and non-breast cancer cell lines. (A and C) RNA obtained from various cell lines was either undiluted or diluted 10-fold and used for reverse transcription. SMAR1, cyclin D1, and β-actin (more ...) SMAR1 represses cyclin D1 gene expression.
To address the role of SMAR1 in regulation of cyclin D1, we designed siRNAs corresponding to the sequence of SMAR1 as described in Materials and Methods. A scrambled siRNA sequence was used as a negative control. In the presence of 200 nM SMAR1 siRNA (RS), there was a complete knockdown of SMAR1 transcript while no change was found with control siRNA-treated samples (Fig. ). To further validate the results, another set of siRNA was used, siRNA (NS), at the effective concentration of 200 nM. The 293 cells were treated with 150 and 200 nM concentrations of SMAR1 siRNA (RS), SMAR1 siRNA (NS), and control siRNA for 24 h. Cells treated with siRNA or control siRNA were harvested to make total RNA and protein lysate. cDNA obtained from total RNA was subjected to PCR amplification for SMAR1, cyclin D1, and β-actin. As shown in Fig. (left and right panels), downregulation of SMAR1 transcript in siRNA-treated samples was correlated with the increased cyclin D1 transcript. Since both the siRNAs targeted to SMAR1 showed increased expression of cyclin D1, further studies were carried out using SMAR1 siRNA (RS). Similarly, protein levels were verified by Western blot analysis (Fig. , middle panel) where an increase in the expression of cyclin D1 was observed. β-Actin was amplified as a control in all the reactions. Thus, the cell line and siRNA data, where a lowered expression of SMAR1 was observed in relation to increased cyclin D1 expression, collectively suggest the role of endogenous SMAR1 in cyclin D1 regulation. We next examined the status of cyclin D1, cyclin D3, and pRb s807/811 upon treatment with SMAR1 siRNA. No inhibition of cyclin D1 and D3 and pRb was observed in SMAR1-overexpressing siRNA-treated cells (Fig. ).
FIG. 3. SMAR1 affects cyclin D1 transcription, and SMAR1 siRNA reverses repression. (A) Optimization of SMAR1 and scrambled siRNA in MCF-7 cells by RT-PCR analysis. (B) Knockdown of endogenous SMAR1 induces cyclin D1 expression. The left-hand panel shows RT-PCR (more ...)
To verify the transcriptional regulatory effect of SMAR1, a time course experiment overexpressing SMAR1 in MCF-7 cells was done. Downregulation of cyclin D1 transcript was observed from 12 h and peaked at 24 h (Fig. ). No detectable product could be seen at 24 h; however, a very small amount of product reappeared at 36 h. To further validate observations of RT-PCR, a real-time RT-PCR analysis was performed. Fortyfold fewer transcripts were observed at 24 h in SMAR1-transfected cells (Fig. ). Both RT-PCR and real-time RT-PCR results were normalized using human β-actin.
The cDNAs obtained from siRNA-treated samples were subjected to RT-PCR analysis to monitor the cyclin D1 transcript profile at various time points. Transfection of SMAR1 siRNA in SMAR1-overexpressed cells reversed the silencing effect of the cyclin D1 transcript at different time points. Real-time PCR analysis was performed from the same cDNA that showed restored transcript levels in siRNA-treated samples. In real-time transcript analysis, cyclin D1 mRNA was elevated by 10-fold at 24 h (Fig. ). These results suggest that SMAR1 is involved in silencing cyclin D1 transcription upon overexpression in MCF-7 cells.
Mapping of the SMAR1 binding site on the cyclin D1 promoter.
To determine whether the cyclin D1 gene is the direct transcriptional target for SMAR1, we studied in vitro binding of SMAR1 on the human cyclin D1 promoter. The approximately 1-kb region with known putative transcription factor binding sites (the region from −66 to −987) of the cyclin D1 promoter was scanned for the SMAR1 binding site in gel shift assays. Either GST or GST-SMAR1 (1 μg) was used for binding studies with 0.5 and 1 μg of poly(dI-dC), using 10 ng of three radiolabeled probes, I, II, and III, in individual reactions (Fig. ). GST-SMAR1 showed a nucleoprotein complex with probe II against the GST control, while no detectable complexes could be seen with probes I and III (Fig. ). The affinity and specificity of the binding was then documented by cold competition experiments. One-hundred-fold-molar-excess competitor DNA was used for binding reactions. MARβ, a previously known SMAR1 binding sequence, was used as competitor DNA, while a 250-bp fragment obtained from an SK+ vector was used as nonspecific competitor DNA. Unlabeled specific competitor DNA in the binding reaction depleted the complex with GST-SMAR1, while no interference was observed using nonspecific DNA (Fig. ). As reported earlier, the DNA binding domain of SMAR1(350-548) showed the band shift with probe II. However, no significant complex formation was detected with the protein-interacting domain SMAR1(160-350) (Fig. ).
FIG. 4. SMAR1 binds to the cyclin D1 promoter. (A) Schematic representation of the cyclin D1 promoter in which arrows indicate the primers used for amplification of probes. The solid box (probe IV) indicates the 50-bp MAR-like sequence in the cyclin D1 promoter. (more ...)
SMAR1 is a MAR binding protein, and we searched for putative MAR-like sequences in the cyclin D1 promoter. A search using a MAR finder program showed a potential MAR-like sequence in the probe II region (Fig. ). We then designed the oligonucleotides for a 50-bp AT-rich sequence for EMSA. Binding reactions were performed using annealed 50-bp double-stranded oligonucleotides with either GST or GST-SMAR1. Interestingly, complex formation was observed with GST-SMAR1 with both 0.5 and 1 μg poly(dI-dC) competitor DNA (Fig. ). We plotted the protein saturation curve using 50 ng to 1 μg of GST-SMAR1 protein (see Fig. S2 in the supplemental material). Affinity of GST-SMAR1 to probe IV was documented using a cold probe as a competitor. SMAR1 exhibited high affinity to the 50-bp (probe IV) sequence in vitro (Kd = 0.48 to 1.4 nM) (see Fig. S2 in the supplemental material). Specificity of binding to probe IV (50 bp oligonucleotides) was further checked using another well-known 70-bp IgH MAR and scrambled 50-bp fragments as competitor DNAs. There was inhibition in complex formation upon addition of MARβ DNA; however, the 70-bp IgH MAR and scrambled 50-bp fragments did not compete for binding with the 50-bp cyclin D1 oligonucleotide (Fig. ). Thus, although SMAR1 is an AT-rich binding protein, it did not show binding to IgH MAR but exhibits primary sequence specificity to the probe II region of the cyclin D1 promoter. As seen for probe II, the DNA binding region of SMAR1(350-548) showed binding to probe IV, while no binding of the protein interacting domain of SMAR1(160-350) was observed with probe IV (Fig. ). To analyze the role of AT-rich sequences in SMAR1 binding, ATs were replaced by GCs. Replacement of AT by GC ablated the binding of SMAR1 to the oligonucleotidenucleotide showing the significance of the AT-rich sequence (Fig. ).
SMAR1 represses transcription from the cyclin D1 promoter.
Recent reports on negative regulation of cyclin D1 transcription revealed recruitment of HDAC1 on the cyclin D1 promoter. Thus, we examined if downregulation of the cyclin D1 promoter by SMAR1 occurs by recruitment of corepressor molecules like HDACs. To verify this, the full-length cyclin D1 promoter (from −1745 to +137) with the luciferase reporter gene was transfected in 293 cells. Cells were harvested 24 h posttransfection, a time when cyclin D1 repression occurs, as checked by RT-PCR and Western blot analysis. Overexpression of SMAR1 significantly repressed the transcription by 4.6-fold from the cyclin D1 promoter (Fig. ). Cotransfection of SMAR1 and HDAC1 synergistically downregulated the repression by 7.6-fold, while overexpression of HDAC1 alone did not alter cyclin D1 promoter-driven luciferase activity (data not shown). Further, the activity of SMAR1-mediated repression on the cyclin D1 promoter was analyzed upon inhibition of HDAC activity using trichostatin A. SMAR1-mediated cyclin D1 repression was relieved by TSA treatment, strongly suggesting the requirement of HDAC activity for SMAR1-mediated repression of the cyclin D1 promoter. Overexpression of Flag-SMAR1 is shown by Western blotting using the same lysate (Fig. ).
FIG. 5. SMAR1 represses cyclin D1 gene expression in reporter assays. (A) Cyclin D1 promoter activity was checked in 293 cells by luciferase reporter assay. Cotransfections of either Flag-SMAR1 or HDAC1 were done along with cyclin D1 luciferase (CD1 Luc) vector. (more ...)
The results of SMAR1-mediated cyclin D1 repression were verified by employing siRNAs specific to SMAR1. As shown in Fig. , endogenous knockdown of SMAR1 increased the transcriptional activity from the cyclin D1 promoter while scrambled siRNA did not affect the transcription. Overexpression of SMAR1 along with siRNA treatment partially rescued the repression that was caused by SMAR1 (1.4-fold repression compared to 4.6-fold repression caused by SMAR1) on the cyclin D1 promoter, suggesting the role of SMAR1 in cyclin D1 promoter downregulation. The level of SMAR1 expression is shown in Fig. .
Data from EMSA studies indicated the significance of the probe II region for binding of SMAR1 on the cyclin D1 promoter, thus, we made a deletion construct in which the probe II region was deleted from the full-length cyclin D1 promoter (cyclin D1 mut luc). Cotransfection of SMAR1 or HDAC1 with the cyclin D1 mut luc construct did not show any downregulation of luciferase activity (Fig. ). The level of SMAR1 was monitored using Western blotting (Fig. ). These results collectively indicate the significance of SMAR1-mediated repression of the cyclin D1 promoter.
SMAR1 associates with HDAC1, SIN3, and pocket Rbs.
It has been suggested that histone deacetylation could be one of the mechanisms by which repressor proteins (25
) mediate transcriptional repression. First we analyzed the effect of TSA in SMAR1-mediated cyclin D1 repression. Flag-SMAR1 was overexpressed in MCF-7 cells following TSA treatment. As seen in Fig. (lane 2), the repressive effect of SMAR1 was eliminated upon TSA treatment. To get further insights into SMAR1-mediated repression, we studied the association of SMAR1 with specific HDACs. In the in vitro interaction studies, GST-SMAR1 was immobilized on GST beads, and 300 μg of 293 cell lysate was incubated for 10 h to check interaction with HDAC1. As shown in Fig. , endogenous HDAC1 interacts with GST-SMAR1. In a similar experiment, overexpressed Flag-HDAC1 was immobilized on Flag beads to which in vitro-translated [35
S]methionine-labeled SMAR1 was added and incubated for 4 h. Detection of 35
S-labeled SMAR1 in Flag-HDAC1-pulled samples further confirmed the in vitro interaction between SMAR1 and HDAC1 (Fig. ). Domain specificity of SMAR1 interaction with HDAC1 was delineated using GST-SMAR1(160-350) and GST-SMAR1(350-548). As shown in Fig. , the protein-interacting domain GST-SMAR1(160-350) showed interaction with HDAC1 while GST-SMAR1(350-548) did not support the interaction.
FIG. 6. SMAR1 interacts with HDAC1, SIN3, and pocket Rbs. (A) MCF-7 cells transiently transfected with Flag-SMAR1 were treated with TSA 48 h posttransfection. Addition of TSA reversed the repression of cyclin D1 (lane 2). (B) 293 cell extract was incubated with (more ...)
To analyze whether SMAR1 directly interacts with HDAC1, coimmunoprecipitation studies were done using 293 lysate or 293 lysate overexpressing Flag-SMAR1. As shown in Fig. (IP: α-SMAR1 for endogenous interaction and IP: α-Flag upon overexpression) and E (IP: α-HDAC1 for endogenous interaction), SMAR1 associated with HDAC1 in endogenous as well as in overexpressed conditions. Domain specificity of SMAR1 and HDAC1 interaction was performed by immunoprecipitations upon transfection of Flag-tagged SMAR1 truncations SMAR1(160-350) and SMAR1(350-548). Consistent with GST pulldown assay data, SMAR1(160-350) showed interaction with HDAC1 (Fig. , middle panel).
To assess the corepressor complex associated with SMAR1 and HDAC1, coimmunoprecipitations were carried out using 293 lysate or 293 lysate that overexpresses Flag-SMAR1. SMAR1- or Flag-immunoprecipitated samples revealed the presence of endogenous mSin3A and mSin3B proteins, as also shown in GST pulldown assays (Fig. , IP: α-SMAR1 for endogenous interaction and IP: α-Flag upon overexpression). The specificity of the interaction in every immunoprecipitation is shown by IgG controls (preimmune control). To further examine whether SMAR1, HDAC1, Sin3A, and Sin3B could form the ternary complex, we performed two-step coimmunoprecipitation studies using 293 cell lysate. In the first immunoprecipitation reaction, SMAR1 antibody was used to pull SMAR1, and the associated complex was eluted in Laemmli buffer. The eluate was then immunoprecipitated with either HDAC1 antibody or control IgG, followed by Western blot analysis to detect Sin3A and Sin3B. As shown in Fig. (right panel), Sin3A and Sin3B were present in the final immunoprecipitate but not in the control immunoprecipitate, indicating SMAR1, HDAC1, Sin3A, and Sin3B exist as a complex. These results, along with immunoaffinity purification results (see Fig. S3 in the supplemental material), indicate SMAR1 is associated with the HDAC1/SIN3 complex. Further nuclear colocalization of SMAR1, HDAC1, Sin3A, and Sin3B were visualized using confocal studies (see Fig. S4 in the supplemental material).
Tumor suppressor Rb regulates the transcriptional events important for cell proliferation. Withdrawal of the cell cycle due to inhibition of E2F-regulated genes was observed upon binding of pRb to E2F species. pRb, along with related pocket proteins p107 and p130, utilizes different mechanisms to elicit this effect. GST pull-down assays and immunoprecipitations showed the interaction of SMAR1 with p107 and p130 (Fig. ). These results suggest that SMAR1 forms a multiprotein complex by associating with HDAC1, SIN3, and pocket Rbs.
SMAR1 associates with the corepressor complex at the cyclin D1 promoter locus.
To determine if SMAR1 is directly recruited to the cyclin D1 promoter in vivo and its correlation to the HDAC1 recruitment, we performed ChIP assays in 293 cells and MCF-7 cells. As shown in Fig. (left panel), the recruitment of both SMAR1 and HDAC1 along with Sin3A/Sin3B was observed on the probe II region in 293 cells under endogenous conditions, suggesting the occupancy of the cyclin D1 promoter by SMAR1. However, much less amplification of probe II was observed in p107- and p130-immunoprecipitated DNA. In a similar experiment of chromatin immunoprecipitation using SMAR1 and HDAC1 antibody in MCF-7 cells, there was no amplification of the probe II region from immunoprecipitated DNA in endogenous conditions (Fig. , right panel). Lack of probe II amplification in MCF-7 cells in endogenous conditions is attributed to undetectable levels of SMAR1 protein in this cell line (as shown in Fig. ). Thus, we overexpressed SMAR1 in MCF-7 cells and studied the recruitment of SMAR1 and the associated HDAC1 protein complex on probe II and probe III regions of the cyclin D1 promoter. As shown in Fig. (right panel), recruitment of SMAR1 and HDAC1 was observed upon overexpression of SMAR1. SMAR1-immunoprecipitated DNA at different time points of transfections from 293 and MCF-7 cells were subjected to PCR amplification using primers designed for probe II and probe III regions. Interestingly, time-dependent recruitment of SMAR1 was observed at the probe II region of the cyclin D1 promoter (Fig. ), while there was no amplification of probe III (data not shown), which was correlated to EMSA results. In both the cell lines, binding of SMAR1 peaked at 24 h to 48 h. Since 293 cells express significant levels of SMAR1 protein, we could see the endogenous levels of SMAR1 occupying probe II at 0 h, while no endogenous SMAR1 was observed in MCF-7 (Fig. ). These results thus indicated the time-dependent recruitment of SMAR1 (upon SMAR1 overexpression) on the cyclin D1 promoter that peaks at 24 h, which is correlated with drastic downregulation of transcript and protein levels. To check for the functionality of SMAR1 with HDAC1, SIN3, and pocket Rbs on the cyclin D1 promoter, ChIP assays were performed. Immunoprecipitated DNA samples from 293 and MCF-7 at the 24-h time point were subjected to PCR using primers for probe II and probe III. We found specific interaction of HDAC1, Sin3A, Sin3B, p107, and p130 on probe II (Fig. ). Amplification of probe III was seen with p107- and p130-pulled fractions, while HDAC1-pulled fractions failed to show amplification. Phospho-Stat and E2F1 transcription factor binding on the cyclin D1 promoter was also studied. A low amplification of probe II was observed in E2F1-pulled DNA, while no amplification could be detected for p-Stat5 (Fig. , left and right panels). As a positive control, input DNA was used while nonspecific amplification was monitored by using mouse IgG-, rabbit IgG-, and no-antibody-pulled DNA samples. These results are consistent with the direct recruitment of HDAC1, SIN3, and pocket Rb complexes by SMAR1 on cyclin D1, which is responsible for the observed repressive effects.
FIG. 7. SMAR1 recruits the repressor complex at the cyclin D1 promoter. Chromatin immunoprecipitation was performed as described in Materials and Methods. (A) Endogenous recruitment of SMAR1, HDAC1, Sin3A/Sin3B, p107, and p130 on the cyclin D1 promoter probe (more ...) SMAR1-associated HDAC1 deacetylates histones in vitro.
Since our results demonstrate SMAR1 interaction with HDAC1 on the cyclin D1 promoter, we wanted to analyze the functionality of this association. Samples overexpressing Flag-SMAR1 were subjected to Flag pull-down assays and directly assayed for HDAC1 activity. Labeled histones were incubated with either recombinant HDAC1 or Flag-pulled samples. Upon fluorography, we observed significant HDAC activity associated with the Flag-SMAR1-pulled complex (Fig. ). A positive control using recombinant HDAC1 showed strong deacetylase activity. However, in a reaction of recombinant HDAC1 along with TSA treatment, we did not observe deacetylase activity. The ability of SMAR1-associated complex to deacetylate the histones suggested a possible role of SMAR1 in deacetylating the cyclin D1 promoter.
FIG. 8. Histone modifications at the cyclin D1 promoter locus. (A) HDAC activity was determined in Flag-immunoprecipitated fractions obtained from Flag or Flag-SMAR1 overexpressed 293 cells upon incubation with labeled histones. (B) In ChIP assays, chromatin (more ...) SMAR1-recruited complex deacetylates histones in vivo.
Since SMAR1-associated complex deacetylated the acetylated histones, we monitored the acetylation status of the cyclin D1 promoter by ChIP analysis using acetylated H3K9 and H4K8 antibodies. We observed that probe II chromatin was acetylated both at H3K9 and H4K8 in mock- compared to SMAR1-transfected cells, and the ratio of deacetylation varied by threefold at 24 h (Fig. ). H3 Ser-10 has been shown to be phosphorylated upon K9 acetylation in active chromatin. In SMAR1-overexpressed cells, the phosphorylation status was reduced by threefold at 24 h (Fig. , middle panel). To check whether this effect was restricted to probe II, where SMAR1 and HDAC1 are recruited, the probe III region was also analyzed (Fig. ). A similar status of deacetylation was observed with probe III, suggesting deacetylation is not restricted to the probe II region.
Endogenous depletion of SMAR1 increased acetylation of histones at the cyclin D1 promoter.
Since the expression of cyclin D1 was increased in 293 cells upon knockdown of endogenous SMAR1, we further verified the endogenous SMAR1-mediated recruitment of HDAC1, SIN3, p107, and p130 to the cyclin D1 promoter by using siRNA specific for SMAR1. We depleted the endogenous SMAR1 from 293 cells using siRNA. As shown in Fig. , we do not observe either SMAR1, HDAC1, Sin3A/Sin3B, p107, or p130 recruitment on probe II upon siRNA treatment, while cells treated with scrambled siRNA showed recruitment of both SMAR1 and HDAC1 on the probe II region. The SMAR1 binding site MARβ was amplified from the same set of samples that served as positive controls (Fig. ). We further analyzed the status of histone acetylation on the cyclin D1 promoter in siRNA-treated and -untreated cells using acetyl-H3K9 and -H4K9 antibodies. Acetylation of H3K9 and H4K8 was increased by twofold in siRNA treatment compared to the scrambled fragments on the probe II and III region of the cyclin D1 promoter, indicating the reversal of deacetylation mediated by endogenous SMAR1 (Fig. ).
SMAR1 directs the histone modifications at a distance.
After examining the role of SMAR1 in histone modifications at the cyclin D1 promoter region (both on probes II and III), we next analyzed whether SMAR1 directs the chromatin remodeling at a distance. Our studies mapped SMAR1 binding to a MAR-like consensus (probe II) in the cyclin D1 promoter region, thus, we searched for a putative MAR consensus 10 kb upstream of the cyclin D1 promoter. Although the EMSA studies have shown that SMAR1 specifically binds and exhibits high affinity (Kd = 0.48 to 1.4 nM) to probe IV in the cyclin D1 promoter, we further studied whether SMAR1 has any additional binding site on the MAR-like consensus observed in the nearby region of the cyclin D1 promoter. We designed a set of primers for ChIP studies of 3 kb, where there was no MAR consensus, and of the 5 kb upstream of the promoter, where we observed a MAR-like region (designated probes VI and VII, respectively). Chromatin immunoprecipitation using SMAR1 antibody in 293 cells revealed no amplification of the probe VI and VII region, suggesting that SMAR1 does not have an additional binding site and specifically binds to probe II (Fig. , left panels). ChIP was done using three sets of primers spanning 1-kb regions around the MAR stretch observed, and the results were consistent with the probe VII data represented in Fig. . We then studied the recruitment of HDAC1, Sin3A, and Sin3B in the probe VI and VII region. As shown in Fig. (middle panels), we did not observe recruitment of any of the above-mentioned factors.
Since overexpression of SMAR1 deacetylated the histones in the probe II and III region and depletion of SMAR1 increased acetylation in this region, we studied whether SMAR1 controls the histone acetylation status at a distance. Therefore, we immunoprecipitated chromatin from 293 cells and 293 cells overexpressing SMAR1 (24 h) using acetyl-H3K9 and acetyl-H4K8 antibodies. Decreased amplification of the probe VI and VII region in SMAR1-overexpressing cells indicated deacetylation of the probe VI and VII region compared to 293 cells alone (Fig. , respectively, right panels).
To further prove the role of SMAR1 in modifying histones, at a 5-kb distance ChIP assays were performed upon depletion of endogenous SMAR1. Depletion of endogenous SMAR1 in 293 cells showed an increased amplification of the probe VII region by twofold, reflecting an increased acetylation. These results suggest that the SMAR1-mediated repressive effect spreads over at least the 5-kb region that has been studied (Fig. ).