The −274 to −237 segment of the FRA-1 containing a c-FOS SRE-like sequence is required for TPA and EGF-inducible transcription
We found that the −379 to +32 bp 5′-flanking region (Figure 1a) mediated a high level of TPA-inducible FRA-1 promoter activity in human pulmonary (A549 and 1HAEo) and breast (MCF-7) epithelial cell lines, respectively, as compared to the −283 to +32 bp segment. Recently, we have shown that the −318 TRE of the FRA-1 promoter is necessary for its TPA-inducible expression in A549 cells (Adiseshaiah et al., 2003). We have shown that c-JUN and FRA-2 proteins are recruited to the endogenous FRA-1 promoter following TPA stimulation. In the present study, we examined whether the promoter region encompassing −318 TRE alone could confer the mitogenic response to a heterologous promoter. The −379/−269 bp fragment of the FRA-1 promoter was cloned upstream of the SV-40 early promoter in the pGL3-promoter vector, to generate −379/−269 SV-Luc (Figure 1b). Cells were transfected with −379/−269 SV-Luc, serum-starved and then stimulated with DMSO, TPA or EGF. The basal activity of the −379/−269 SV-Luc was similar to that of parent vector SV-Luc. Interestingly, TPA, EGF and serum (data not shown) failed to stimulate the −379/−269 SV-Luc beyond its basal level, suggesting a requirement for additional cis-element(s) to confer mitogen-induced response.
We next examined whether inclusion of a DNA segment located between −276 and −239 could restore mitogen inducibility to the promoter. We chose this region because it exhibited a high homology (see below) to the SRE of c-FOS. Indeed, inclusion of this DNA segment restored TPA, EGF and serum (data not shown) response to the −379/−269-SV-Luc (Figure 1b). These results indicate that the −379/−237 region is sufficient for high-level TPA and EGF-inducible FRA-1 transcription. To demonstrate the importance of the −379/−269 enhancer fragment, we generated the −379/−237 SV-Luc construct bearing mutations in the −318 TRE and analysed the promoter activity in response to TPA, EGF and serum. Mutation of this site drastically reduced TPA, EGF and serum (data not shown) inducibility. Thus, the −318 TRE is critical for mitogen-sensitivity even in the context of a neutral promoter.
The −379 to −237 enhancer elements are conserved across species
Alignment of the human, mouse and rat promoters revealed that several crucial elements are conserved across species (Figure 2a). In particular, we found that the −276 to −239 bp segment of FRA-1 contained a putative ATF site, and a TCF site and the CArG box (hereafter referred to as the, SRE). Sequence analysis also indicated that the positions and orders of the −318 TRE, −343 GC box, and −377 EBS (Ets-binding site) were highly conserved (Figure 2a). We next compared the SREs of the members of the FOS family. Putative SREs were well conserved among all members of the FOS family. The SREs and ATF sites of all FOS family members are separated by five nucleotides (Figure 2b). However, the distances between the TCF site and the CArG box are different for FRA-1 (6 bp) and c-FOS (2 bp). Whether this difference accounts for the distinct kinetics of expression of c-FOS and FRA-1 is unclear.
Mutations in the SRE and ATF sites suppress FRA-1 promoter activity
To assess the contribution of putative SRE and the ATF sites to the regulation of FRA-1 transcription in response to mitogenic signals, we generated additional promoter mutants lacking the TCF, CArG and ATF sites in the context of −379 to + 32 fragment (Figure 3a), and their impact on TPA and EGF induced expression was analysed. In response to TPA and EGF, a robust induction in the promoter activity was noticed with the wild-type promoter. However, mutation of the individual TCF site (AGGAA to AgTAc), the CArG box (CCATGTATGG to CCATGTTca), or the ATF site (GCTACGTCA to GgTAtGTtA) significantly diminished (>50%) both TPA and EGF-induced expressions of the reporter. These findings suggest that SRE and the ATF sites play an important role in regulating mitogen inducible FRA-1 transcription. Mutations within the TCF, CArG and ATF sites in the context of a heterologous (SV-40) minimal promoter also similarly reduced both TPA- and EGF-inducible FRA-1 transcription (data not shown). Collectively, these results suggest that SRE and the ATF site contribute to the induction of FRA-1 by mitogens.
Analysis of transcription factor binding to the −276/−239 bp FRA-1 promoter
Based on the above studies, we chose a 38-bp DNA segment (−276/−239 region) containing the SRE (the TCF site and the CArG box), and the ATF site (see Figure 4a for sequence) for use in electrophoretic mobility shift assay (EMSAs) to identify protein factors that bind to it following TPA stimulation. The TCF-CArG-ATF oligo formed three protein complexes, designated I, II and III. The intensity of these complexes was enhanced following TPA (Figure 4b, compare lanes 1 and 2). The specificity of these complexes was analysed by competition assays using the unlabeled TCF-CArG-ATF (self) and other oligos: consensus TRE, consensus GC-box and the ATF oligo (see Figure 4a for sequence). Incubation of nuclear extracts with the unlabeled ‘self’ oligo markedly reduced the formation of all three complexes (compare lanes 3 and 4 to lane 2). c-FOS SRE (lane 5) or FRA-1 SRE (lane 6) lacking the ATF site completely blocked the formation of complex I. Although consensus TRE (lane 7) significantly diminished the formation of complex II, it had no such effect on complex I formation. In contrast, the consensus GC box had no appreciable effect on any of the three complexes (lane 8). To determine the specificity of ATF protein binding to the FRA-1 promoter, competition assays were performed using the unlabeled ATF site of the FRA-1 promoter (Figure 4b, lanes 9–12). The FRA-1 ATF oligo strongly reduced the formation of complex II in a concentration-dependent manner. Collectively, these observations suggest that the TCF, SRF and ATF-like proteins bind to the −276/−239 region.
We next used antibodies specific to various transcription factors to characterize the nature of the protein complexes binding to the TCF-CArG-ATF sites of the FRA-1 promoter (Figure 4c). In unstimulated (−) and TPA-stimulated (+) A549 and MCF cells, the anti-SRF antibody ‘supershifted’ complex I (Figure 4c, lanes 2 and 6). Note that in TPA-stimulated cells, the binding of SRF (lane 6) was slightly higher than in the control (lane 2). The anti-Elk1 antibody specifically decreased the formation of complex II in untreated (lane 3) and TPA-stimulated (lane 7) cells, while anti-SAP1 antibody caused no appreciable effect (lanes 4 and 8). To better visualize supershifted and blocked DNA–protein complexes formed with nuclear extracts of A549 cells, EMSA gels were exposed for a longer and a shorter time, respectively (Figure 4c, left panel). Similar result was obtained with the nuclear extracts isolated from MCF-7 cells (Figure 4c, right panel). As shown in Figure 4d, both anti-ATF1 (lanes 2 and 4) and anti-CREB (lanes 7 and 8) specific antibodies supershifted the complex II. In contrast, the anti-ATF2 and anti-ATF4 antibodies had no such effect (data not shown). Similar results were obtained in EMSAs with the nuclear extracts isolated from unstimulated and TPA-stimulated MCF-7 cells (Figure 4d, right panel, lanes 9–16). Incubation of labeled TCF-CArG-ATF probe with anti-SRF (lane 17), anti-ATF1 (lane 18), or anti-CREB (lane 19) antibodies caused no supershift of the probe.
To further confirm the binding of above transcription factors, we have used oligos containing the TCF-CArG or SRE (Figure 5a) and the ATF (Figure 5b) sites of the FRA-1 promoter as probes in EMSAs. As seen with a full-length probe, incubation of nuclear extracts with the TCF-CArG probe yielded three specific complexes, I, II and III. The addition of anti-SRF completely shifted the complex I, while it significantly blocked the TCF complex II in A549 (lanes 3 and 4) and MCF-7 (lanes 12 and 13) cells. When incubated with the probe alone SRF antibody yielded no such supershift (Figure 5a, lane1). Anti-Elk1 antibody specifically decreased the complex II formation both in A549 (lanes 6 and 7) and MCF-7 (lane 14 and 15) cells. Anti-SAP1 antibody (lanes 16 and 17) also had a similar effect both in A549 (data not shown) and MCF-7 (Figure 5a, lanes 16 and 17) cells. Upon incubation of nuclear extracts with the ATF site formation of a single specific complex was noted (Figure 5b). The anti-ATF1 antibodies (lanes 2 and 5) markedly blocked the formation of this complex, while the CREB antibodies (lanes 3 and 6) supershifted the complex in nuclear extracts isolated from control and TPA-treated A549 cells. ATF1 and CREB antibodies used in the present study do not crossreactive with other ATF/CREB transcription factors. Taken together, these results (Figures 4 and 5) indicate that TCFs, such as Elk1, SRF, ATF1 and CREB transcription factors, appear to bind to the promoter of FRA-1 in unstimulated cells, and TPA stimulation has a minimal effect on the binding.
The FRA-1 promoter is occupied by SRF, Elk1, CREB and ATF proteins in vivo
Although the EMSA results suggest the engagement of Elk1, SRF, ATF1 and CREB with the enhancer elements derived from the FRA-1 promoter, they do not directly address whether the endogenous FRA-1 promoter recruits these factors or not. We have shown earlier the recruitment of c-Jun to the endogenous FRA-1 promoter in response to TPA treatment (Adiseshaiah et al., 2003). To determine whether SRF, Elk1, CREB and ATF1 are similarly recruited to the promoter in vivo, we performed a ChIP assay using the crosslinked chromatin fragments prepared from control and TPA-stimulated A549 cells (Figure 6). In separate reactions, chromatin was immunoprecipitated using the anti-SRF, anti-Elk1, anti-CREB or anti-ATF1 antibodies. Nonimmune IgG was used as a negative control for these studies (Figure 6). We also used a positive control IP reaction with anti-c-Jun-antibody in parallel to demonstrate the recruitment of c-Jun. DNAs eluted from the immunoprecipitated chromatin fragments were PCR amplified using FRA-1 promoter-specific primers. As expected, ChIP assays using the nonimmune IgG showed no amplification of the FRA-1 promoter. The SRF, Elk1, CREB and ATF1 proteins were present in the basal transcriptional complex formed with the FRA-1 promoter in the unstimulated state (lanes 1–3). TPA treatment did not significantly enhance the recruitment of these proteins to the promoter (lanes 4–6). This finding is consistent with previous reports that have shown a constitutive occupation of SRF and Elk1 at the c-fos SRE (Herrera et al., 1989; Konig, 1991; Runkel et al., 1991; Tornaletti and Pfeifer, 1995). In contrast, there was a significant increase in c-Jun recruitment to the FRA-1 promoter after TPA stimulation (lanes 4–6), as compared with unstimulated cells (lanes 1–3).
SRF, Elk1, ATF1 and CREB regulate TPA-inducible FRA-1 transcription
To support the physical interaction studies (ChIP and EMSA), we next determined the transcription activating effects of SRF, Elk1, ATF1 and CREB. Previous studies (Brusselbach et al., 1995; Schreiber et al., 1997), including ours (Adiseshaiah et al., 2003), have reported a critical role for c-Jun in mediating the induction of FRA-1 by mitogenic stimuli. However, the effects of SRF and TCFs, and ATF1 and CREB in mediating FRA-1 transcription by extracellular stimuli have not yet been determined. Therefore, we have examined their roles in mediating FRA-1 induction by TPA. We have used expression vectors coding for dominant-negative mutants of SRF, Elk1, ATF1, ATF4 and CREB proteins. Coexpression of an SRF mutant (Lee et al., 1992) significantly suppressed TPA-induced FRA-1 promoter activity (Figure 7a). Similarly, an Elk1 mutant (Yang et al., 1998) and an ATF1 mutant (Gupta and Prywes, 2002) markedly diminished TPA-inducible expression of the reporters (Figure 7b). A mutant form of CREB protein also inhibited TPA-inducible expression of the reporter (Figure 7c). In contrast, coexpression of ATF4-mt did not suppress TPA induced FRA-1 promoter activity (Figure 7d). Collectively, these results suggest that SRF and TCF proteins, such as Elk1, ATF1 and CREB proteins regulate TPA-inducible FRA-1 expression.
Silencing of SRF, Elk1 and c-JUN expression significantly suppresses TPA-stimulated FRA-1 promoter activity
To examine whether attenuation of SRF, Elk1 and c-JUN expression alters FRA-1 promoter activity, A549 cells were transfected with the 379-Luc promoter reporter construct along with 20 nM of siRNAs specific for each of these transcription factors. After 36 h, cells were serum-starved for 12 h prior to TPA stimulation and luciferase activity was measured. We have chosen 20 nM siRNA in our experiments (Figure 8a) although it suppressed ∼50% of the total levels of SRF, Elk1 and c-JUN proteins (data not shown). We could not escalate the dose of these siRNAs in these experiments because at higher concentrations (50–100 nM) these siRNAs exhibited nonspecific inhibitory effects (data not shown). Both SRF (Figure 8a, top panel, lanes 5 and 6) and Elk1 (Figure 8a, bottom panel, lanes 5 and 6) siRNAs specifically reduced the SRF and Elk1 protein levels as compared to transfection reagent control (RC, lanes 1 and 2) and the negative control siRNA (NCsi, lanes 3 and 4). The siRNAs exerted no discernible inhibitory effect on the expression of ERK2 protein (internal control). As shown in Figure 8b, incubation of A549 cells in the presence of Elk1, SRF or c-JUN siRNAs caused a significant (P-values <0.002, <0.001, and <0.002, respectively) decrease in the TPA-stimulated FRA-1 promoter activity as compared to the NCsi (Figure 8b, bar 2). However, no appreciable difference was detectable between transfection reagent (bar 1) and NCsi (bar 2) transfected cells. These results suggest a role for SRF, Elk1 and c-JUN in TPA-induced FRA-1 transcription.
Overexpression of SRF mutant suppresses c-JUN stimulated FRA-1 promoter activity
We have recently reported that c-Jun-enhanced the induction of FRA-1 is mediated in part through TRE located at −318 (Adiseshaiah et al., 2003). We therefore investigated whether SRE is required for this c-Jun-enhanced FRA-1 transcription. To examine this aspect, we transfected cells with an srf mutant along with the 379-Luc and pRK-TL plasmids in the presence of an empty or a c-jun expression vector. As expected, c-Jun markedly stimulated reporter upon coexpression. However, mutant SRF protein significantly suppressed c-Jun-enhanced FRA-1 promoter activity (Figure 9a). In contrast, coexpression of a mutant form of ATF4 transcription factor along with c-Jun did not inhibit the FRA-1 promoter activity. The disruption of the individual TCF, CArG box, and ATF sites, located at position −274, −263, and −248, respectively, also strongly inhibited the c-Jun-enhanced reporter expression (data not shown). Together, these observations and the data presented in Figure 3 suggest a requirement for the SRE and ATF sites for a maximal stimulation of the FRA-1 promoter by c-Jun and mitogens, such as TPA.
The −379 to +32 bp 5′-flanking region of FRA-1 is sufficient for a temporal regulation of the reporter like the endogenous gene
To define whether −379 to +32 bp region of FRA-1 promoter construct recapitulates the regulatory profile of the endogenous gene, cells were transfected with the 379-Luc and pRK-TL plasmid constructs, serum-starved for 24 h, and then stimulated with TPA. Lysates were prepared after 0, 15, 30, 60 or 90 min of stimulation and luciferase activity was determined (Figure 10a). No significant change in the promoter activity occurred up to 30-min post-TPA stimulation. In contrast, FRA-1 promoter activity is nearly four-fold higher by 60 min and remained high until 180 min.
To analyse the endogenous FRA-1 and c-FOS expression, cell cultures grown in parallel were treated with TPA for 0–90 min, total RNA was isolated, and the endogenous mRNA levels were analysed by real-time PCR (Figure 10b). As anticipated, TPA maximally stimulated the c-FOS mRNA expression by 30 min, as compared to the ‘0’ min control (right panel). In contrast, no significant change in the levels of FRA-1 mRNA was noticed at this time point (left panel). However, FRA-1 mRNA was maximally expressed at 90 min after TPA stimulation (bar 5, left panel), whereas c-FOS expression returned to basal level at this time point (bar 5, right panel). Together, these results show that the -379 to +32 bp fragment of FRA-1 promoter contains sufficient necessary elements that temporally mimic the induction profile of the endogenous gene.
Dr. Malcolm S. Adiseshiah Centenary Celebrations
(April 2009 - April 2010)
MIDS is celebrating the Centenary of its Founder, Prof.Malcolm S.Adiseshiah, during April 2009 - April 2010. During this Centenary year, MIDS is planning to organize a series of academic events and publications in reverence to the memory of its Founder. The Centenary Year started with a Founder-s Day Lecture: "Lineages of Political Society" by Prof.Partha Chatterjee (Centre for Studies in Social Sciences, Kolkata) on April 3, 2009. The Centenary celebration was formally launched in May 4, 2009 by Prof.M.S.Swaminathan (Chairman of MSSRF and Member of Parliament) with his ceremonial presentation: "Agrarian Crisis: The way ahead". On the same day, the launch function was also followed by a MIDS-NABARD co-sponsored Workshop on: "Financing for Rural Development". In this workshop, a number of scholars and experts have presented papers and participated in the deliberations. They include: Dr.S.K.Mitra (NABARD, Mumbai), Dr.K.V.Raghavulu (NABARD, Chennai), Prof.R.S.Deshpande (ISEC, Bangalore) Prof.Srijit Mishra (IGIDR, Mumbai), Prof.K.Nagaraj (MIDS, Chennai), Prof.D.Narasimha Reddy (University of Hyderabad, Hyderabad), and Prof.K.Narayanan Nair (CDS, Trivandrum).
During the Centenary year MIDS is also planning a number of public lectures under `Prof.Malcolm Adiseshiah Centenary Public Lecture Series-, which will be organized jointly with a number of prominent academic institutions both in Chennai and in other major educational centres in Tamil Nadu. The First lecture under this series was delivered by Prof.Kaliappa Kalirajan (Australian National University, Canberra) on July 9, 2009 at MIDS. The title of his lecture was: "Globalisation: An Analysis of Key Aspects of the Emergence of Big Malls and Retails in India". Two more such Public lectures were also organized, one at the Department of Economics, Annamalai University and another at the Kandaswamy Kandar College near Namakkal. Besides several publications being planned during the Centenary year, efforts are also afoot to release a commemorative stamp for Prof.Adiseshiah. In addition to MIDS, other organizations are also planning events to celebrate the Centenary of Prof. Malcolm Adiseshiah. For instance, the University of Madras, where Prof.Adiseshiah has been a Vice Chancellor, is organizing special lecture series under which a number of prominent persons are invited to deliver special lecture. Similarly, the Institute of Development Studies at the Mysore University is also planning a joint event with MIDS and ISEC, which will be held early next year.
The Faculty and Staff at MIDS hope these academic events and publications will go a long way both in cultivating the memory of Prof.Adiseshiah as well as to help inspiring many more such selfless individuals with a immense contribution to the academic and socio-economic development of Tamil Nadu in particular and India in general.