Figure 3. Internally initiated Sp3-derived proteins are karyophilic and form protein-DNA complexes that are indistinguishable from RCE-RCP complex 2. (A) Protein-DNA binding assays. A radiolabeled wild-type c-fos RCE probe was incubated with C-33A nuclear extracts or in vitro translated Sp3, M1 or M2 proteins alone (-) or in the presence of a 400-fold molar excess of unlabeled wild-type (+WT) or mutated (+Mut) RCE oligonucleotides. RCE-RCP complexes are indicated on the left. (B) Western blots of SL2 nuclear extracts prepared from untransfected cells (-) or cells transfected with equivalent amounts of pPacSp3, pPacM2 or pPacSp1 expression vectors. Blots were probed with anti-Sp3 antiserum ([alpha]SP3 TAD, I-5) or with antiserum prepared against Sp1 (Sp1). (C) Protein-DNA binding assay using a wild-type c-fos probe and C-33A nuclear extracts or nuclear extracts prepared from SL2 cells transfected as in (B). RCE-RCP complexes are indicated on the left.

To determine if the 78-80 kDa Sp3-related proteins synthesized in vivo are structurally identical to M1 and M2 proteins synthesized in vitro we wished to compare their partial proteolytic maps following cleavage with reagents such as V8 protease or cyanogen bromide. Unfortunately, such experiments could not be performed due to inefficient precipitation of radiolabeled Sp3- related proteins from cell extracts. To confirm that the 78-80 kDa Sp3-related proteins synthesized in vivo share antigenic determinants with M1, we prepared GST or GST-M1 fusion proteins in bacteria and incubated [alpha]Sp3 TAD antiserum with an excess of these proteins in vitro. Following depletion for reactivity with each of these bacterial proteins, [alpha]Sp3 TAD antiserum was employed to detect endogenous Sp3-related proteins in mammalian cells. As shown in Figure 2 C, preincubation of [alpha]Sp3 TAD antiserum with GST had little or no effect on reactivity with Sp3 or Sp3-related proteins. In contrast, preincubation of [alpha]Sp3 TAD antiserum with GST-M1 results in antiserum that detects the 115 kDa Sp3 protein doublet but not the 78-80 kDa Sp3-related proteins. These data clearly indicate that [alpha]Sp3 TAD antiserum binds antigenic determinants in M1 that are shared by the 78-80 kDa Sp3-related proteins synthesized in vivo. Moreover, these data indicate that the Sp3-related proteins do not carry antigenic determinants bound by [alpha]Sp3 TAD antiserum that are encoded upstream of the M1 translational initiator.

Given that M1 and M2 proteins are likely to be structurally identical to the 78-80 kDa Sp3-related proteins synthesized in vivo, we wished to establish whether M1 and M2 are also functionally equivalent. To determine if the DNA binding activities of M1 and M2 are similar to RCP 2, in vitro translated Sp3, M1 and M2 proteins were employed in protein-DNA binding assays. As shown in Figure 3 A, M1- and M2-derived proteins form protein-DNA complexes that co-migrate with RCP 2 harvested from mammalian nuclear extracts. Moreover, as shown by competition experiments with wild-type (WT) and mutated (Mut) RCE oligonucleotides, the formation of M1 and M2 protein-DNA complexes is dependent on the integrity of nucleotides that are identical to those required for formation of protein-DNA complexes from nuclear extracts. To determine if Sp3 proteins that arise via internal initiation are karyophilic, stably expressed in vivo and capable of binding DNA as expected, we subcloned M1 and M2 cDNAs into expression vectors and transient transfections were performed in parallel with Sp3 and Sp1 using Drosophila SL2 cells as recipients. Nuclear extracts were prepared from transiently transfected SL2 cells and examined in: (i) Western blots for protein expression; (ii) protein-DNA binding assays (Fig. 3 B and C). Each of the transfected expression constructs synthesized nuclear proteins of the predicted size that were detected in Western blots with antisera prepared against the proteins themselves as well as their epitope tag (Fig. 3 B and data not shown). Moreover, each exogenously expressed protein formed protein-DNA complexes of the expected size with radiolabeled RCE oligonucleotides (Fig. 3 C and data not shown). Taken together with the immunochemical evidence cited above (Figs 1 and 2 C), data from protein-DNA binding assays strongly suggest that: (i) the 78-80 kDa Sp3-related proteins detected in vivo are Sp3 isoforms that arise via internal translational initiation at the M1 and M2 methionine residues; (ii) internal initiation within Sp3 mRNA accounts for RCP complex 2. Since we have previously reported that RCP complex 2 appeared to be formed by a protein of 80 kDa (11 ), we presume that the coincident electrophoretic migration of protein-RCE complexes formed by the 78 and 80 kDa Sp3-derived proteins prevented their resolution as a protein doublet following photoaffinity labeling.

Internally initiated isoforms of Sp3 function as potent inhibitors of Sp-mediated transcription in vivo

Since M1 and M2 lack substantial portions of the Sp3 trans- activation domain, we speculated that their potency as trans-activators was likely to be significantly compromised. Furthermore, given that M1/M2 appear to have the same DNA binding specificity as Sp1/Sp3, we hypothesized that co-expression of M1 or M2 with Sp1 or Sp3 might interfere with Sp-mediated transcription. To examine these possibilities we utilized a well-characterized promoter-CAT construct prepared from the hamster DHFR promoter as a reporter gene and Drosophila SL2 cells as recipients to monitor Sp-mediated trans-activation in vivo. We and others have previously shown that Sp1 is a potent trans-activator that induces dose-dependent transcription of DHFR and a variety of additional mammalian promoters in SL2 cells (12 ,16 ,38 ,44 ). We have also shown that Sp3-mediated DHFR transcription approximates that of Sp1 (34 ). Consistent with the supposition that truncation of the Sp3 trans-activation domain might negatively effect M1/M2-mediated transcription, transient expression of M1 or M2 in SL2 cells led to little or no DHFR-mediated transcription above that of the reporter gene alone (Table 1 and data not shown). As might be predicted given their common nucleotide binding specificities, co-expression of M2 with Sp1 or Sp3 resulted in a profound reduction in Sp-mediated DHFR transcription (Table 2 ). Similar results were noted in parallel co-transfection experiments with M1 (data not shown). Interestingly, although Sp1 and Sp3 are expressed at equivalent levels in vivo (Fig. 3 B), it is apparent that Sp1 and Sp3 differ in their relative sensitivity to transcriptional inhibition; Sp3-mediated transcription is at least 10-fold more sensitive to transcriptional inhibition by M1/M2 than is Sp1 (Table 2 ). For example, transfection of 50 or 100 ng M2 with 50 ng Sp3 was sufficient to reduce Sp3-mediated transcription by 74 and 91% respectively. In contrast, 1000 ng M2 were required to diminish Sp1-mediated transcription to equivalent levels. The negative effect of M1 and M2 co-expression on Sp-mediated transcription is not restricted to the DHFR promoter, as similar results were obtained using promoter-CAT constructs dependent on RCEs derived from the c-fos, c-myc and TGF-[beta]1 genes (Table 2 ; 11 ,12 ). To confirm that transcriptional inhibition by M1/M2 is specific and not due to generalized disruption of transcription in vivo, co-transfection experiments with a GAL4-CAT reporter construct and a GAL4-VP16 fusion protein, a potent trans-activator, were performed. Consistent with the notion that Sp3-mediated transcriptional inhibition is specific, co-expression of M1 or M2 with GAL4-VP16 did not have a dose-dependent effect on VP16- mediated transcription (Table 2 and data not shown). A slight increase in VP16-mediated transcription was noted at low input amounts of M1/M2 that was not apparent with increasing concentrations of DNA. We conclude from these results that internally initiated isoforms of Sp3 do not function as stimulatory transcription factors in SL2 cells. Instead, such isoforms can function as potent inhibitors of transcription when co-expressed with Sp1 or Sp3. Furthermore, we conclude that Sp3 is significantly more sensitive to transcriptional inhibition by internally initiated Sp3 isoforms than is Sp1.

Table 1 . Trans-activation of hamster DHFR promoter by Sp-family members^a

Reporter construct	Additional DNA(s)	Relative CAT activity (+-SE)b	nc
DHFR	-	1.0	10
	Sp1 50 ng	130 (31)	14
	Sp3 50 ng	244 (98)	18
	M1 10 ng	1.5 (0.8)	4
	M1 50 ng	1.0 (0.1)	4
	M1 100 ng	1.2 (0.2)	4
	M1 500 ng	2.0 (1.7)	4
	M2 10 ng	3.0 (0.7)	4
	M2 20 ng	3.2 (1.0)	4
	M2 50 ng	1.9 (0.5)	10
	M2 100 ng	2.9 (0.9)	4
	M2 200 ng	3.0 (0.9)	4

^aDrosophila SL2 cells were transiently transfected with a DHFR-CAT reporter construct alone or in conjunction with the indicated amounts of expression constructs for Sp1 (pPacSp1), Sp3 (pPacSp3), M1 (pPacM1) or M2 (pPacM2). Resulting levels of CAT activity were quantified as previously described (12).^bShown are mean values for percentage acetylation (+-SE) of chloramphenicol per A₆₀₀ of 1 [mu]l total cell extract normalized to the amount of activity recorded for the DHFR-CAT reporter construct.^cn is the number of independent plates of transfected cells analyzed.

DISCUSSION

Rb has previously been shown to govern transcription of a subset of growth control genes, such as c-fos, c-myc and TGF-[beta]1, via discrete promoter elements termed RCEs (6 ,7 ). To determine the mechanism(s) by which Rb regulates RCE function, we have sought to identify and characterize nuclear RCE binding proteins in vitro, determine if such proteins are required for RCE activity in vivo and assess the functional consequence of their interaction with Rb. We have previously determined that RCEs are bound in vitro by Sp1 and Sp3, two members of the Sp family of transcription factors, and that RCE-mediated transcription is stimulated by these factors in vivo (12 ,34 ). Moreover, co-expression of Rb and Sp1 or Sp3 leads to marked `superactivation' of Sp1/Sp3-mediated transcription (12 ,34 ). In this report we demonstrate that: (i) Sp3 encodes at least three distinct transcription factors; (ii) each of these Sp3-related proteins binds RCEs in vitro; (ii) two Sp3-related proteins arise via internal translational initiation within Sp3 mRNA; (iv) internally initiated Sp3 isoforms are potent inhibitors of Sp-mediated transcription. Taken together with recent evidence that Sp1 is an alternatively spliced gene (45 ), our results indicate that transcriptional control of RCEs and, by extension, other promoter elements regulated by Sp family members may be significantly more complex than previously suspected.

Table 2 . Inhibition of Sp-mediated, but not VP16-mediated transcription by an internally initiated isoform of Sp3^a

Reporter construct	Trans-activator	Additional DNA	Relative CAT activity (+-SE)b	nc
DHFR	Sp1 50 ng	-	1.0 (0)	14
		M2 10 ng	1.5 (0.3)	8
		M2 50 ng	1.7 (0.4)	10
		M2 100 ng	1.2 (0.2)	14
		M2 250 ng	0.6 (0.1)	2
		M2 500 ng	0.3 (0.1)	4
		M2 1000 ng	0.1 (0)	2
	Sp3 50 ng	-	1.0 (0)	18
		M2 10 ng	0.8 (0.1)	12
		M2 50 ng	0.3 (0.4)	14
		M2 100 ng	0.1 (0)	18
		M2 250 ng	0 (0)	2
		M2 500 ng	0 (0)	4
FOS	Sp3 50 ng	-	1.0 (0)	3
		M2 100 ng	0.2 (0)	3
MYC	Sp3 50 ng	-	1.0 (0)	3
		M2 100 ng	0.2 (0)	3
TGF-[beta]1	Sp3 50 ng	-	1.0 (0)	3
		M2 100 ng	0.2 (0)	3
GAL4	VP16 10 [mu]g	-	1 (0)	4
		M2 50 ng	1.9 (0.1)	2
		M2 100 ng	1.2 (0.1)	4
		M2 500 ng	1.3 (0)	2

^aDrosophila SL2 cells were transiently transfected with DHFR-CAT, FOS-CAT, MYC-CAT, TGF-[beta]1-CAT or GAL4-CAT reporter constructs in conjunction with the indicated amounts of expression constructs for Sp1 (pPacSp1), Sp3 (pPacSp3), M2 (pPacM2) or GAL4-VP16. Resulting levels of CAT activity were quantified as previously described (12).
^bShown are mean values for percentage acetylation (+-SE) of chloramphenicol per A₆₀₀ of 1 [mu]l total cell extract normalized to the amount of activity recorded for DHFR or GAL4 reporter constructs in the absence of M2.
^cn is the number of independent plates of transfected cells analyzed.

We and others have previously reported that Sp3 stimulates transcription in vivo, however, this observation has not proven to be universal (21 ,34 ,42 ,46 -51 ). Using a cDNA isolated by Hagen et al. (35 ), a number of laboratories have reported that Sp3 has little or no capacity to function as a transcriptional trans-activator. Instead, based on a wide variety of transient co-transfection experiments, Sp3 has been reported to function as a repressor of Sp1-mediated transcription. Since it is now apparent that Sp3 encodes at least three distinct proteins in vivo and that internally initiated Sp3 isoforms function as transcriptional inhibitors, it is likely that these once disparate observations may be easily reconciled, i.e. experiments that employ Sp3 expression vectors that preferentially lead to biogenesis of full-length Sp3 protein will score Sp3 as a transcriptional trans-activator while expression vectors that largely yield internally initiated Sp3 isoforms will score Sp3 as a transcriptional repressor. This conclusion appears to be entirely consistent with the sizes of Sp3 proteins expressed following transient transfections with Sp3 cDNAs (34 ,50 ).

Since all Sp3 cDNAs cloned to date are partial cDNAs lacking 5'-untranslated sequences and the extreme N-terminus of Sp3 protein, we can only speculate on the mechanism governing translational initiation within Sp3 mRNA. One possibility is that the translational start site at the 5'-end of the Sp3 message is partially obscured by RNA secondary structure or embedded within a sequence that is weak relative to internal sites of translational initiation. Presumably this arrangement ensures that appropriate levels of stimulatory and inhibitory Sp3 proteins are synthesized in vivo. That a single mRNA can encode multiple functionally distinct transcription factors may be a more common theme for transcriptional regulation that previously realized. Like Sp3, the C/EBP[alpha] and C/EBP[beta] CCAAT/enhancer binding proteins have previously been noted to encode full-length proteins that function as trans-activators as well as internally initiated isoforms that act as inhibitors of C/EBP function (52 -55 ). As for Sp3, internal translational initiation within C/EBP[alpha] and C/EBP[beta] mRNA results in elimination of a significant portion of their respective trans-activation domains. C/EBP[beta] encodes liver-enriched activating (LAP) and inhibiting (LIP) proteins and the LIP/LAP ratio, which has been shown to be developmentally and hormonally regulated, influences transcription of a variety of cellular genes. Although the precise mechanism by which LIP inhibits LAP function remains to be determined, it is likely that LIP dimerizes with LAP via their respective basic region/leucine zipper domains forming transcriptionally inactive complexes that compete with transcriptionally active LAP dimers for occupancy of DNA binding sites. It is worth emphasizing that our results provide little insight into the mechanism(s) by which internally initiated Sp3 isoforms abrogate Sp-mediated transcription. In protein-DNA binding assays we have previously shown that: (i) Sp1 and full-length and internally initiated Sp3 isoforms are equally abundant in extracts prepared from >20 mammalian cell types grown under a variety of growth conditions; (ii) Sp1 and each Sp3 isoform require identical RCE nucleotides for formation of protein-DNA complexes; (iii) on- and off-rate measurements indicate that each protein-RCE complex is equally stable in vitro (A.J.Udvadia and J.M.Horowitz, unpublished observations). These results would suggest that competition for binding site occupancy between internally initiated Sp3 isoforms and Sp3 or Sp1 could account for diminished Sp-mediated transcription. Yet, if binding site competition were the sole mechanism by which Sp-mediated transcription is inhibited, equivalent levels of inhibition of Sp1 and Sp3 would be expected. Instead, Sp3-mediated transcription is reproducibly at least 10-fold more sensitive to inhibition than is transcription directed by Sp1. Given this intriguing observation, we speculate that disparate degrees of protein-protein interactions may account for the differential sensitivities of Sp1 and Sp3 to inhibition by internally initiated isoforms of Sp3. As one possibility in this regard, we note that the D domain of Sp1 has previously been shown to play an important role in its multimerization and this region is strikingly dissimilar in Sp3. Thus, amino acid differences within the D domains of Sp1 and Sp3 may facilitate higher affinity interactions between full-length Sp3 and its internally initiated isoforms. Yet another possibility springs from the recent identification of a `repression domain' immediately upstream of the Sp3 DNA binding domain (56 ). It is conceivable that interactions between Sp3 molecules via this domain may be favored over those involving Sp1 and Sp3 proteins. Once complexed with Sp1 or Sp3, internally initiated Sp3 isoforms may diminish the capacity of multimeric complexes to bind DNA or prevent formation of transcriptionally active protein-DNA complexes. Consistent with the notion that multimerization of Sp proteins is required for transcriptional repression, Sp3-mediated repression has been reported to be dependent on the number of Sp3 binding sites within cellular promoters (51 ).

Although the mechanism by which internally initiated Sp3 isoforms inhibit Sp1/Sp3-mediated transcription remains obscure, we speculate that this mode of regulation likely plays an important role in cell cycle- and signal-induced transcription. Indeed, cell cycle- or signal-induced expression of a variety of genes, including p21^waf1/cip1, p15^INK4B, CYP11A, mdr1 and acetyl-CoA carboxylase, has been mapped to GC-rich promoter elements that bind Sp family members in vitro (18 ,19 ,23 ,57 -60 ). We predict that cell cycle- and signal-induced alterations of the protein and/or DNA binding activities of internally initiated Sp3 isoforms may account in part for regulation of such differentially expressed genes.

ACKNOWLEDGEMENTS

The authors are indebted to members of the Horowitz laboratory, Michael Datto and Xiao-Fan Wang for helpful discussions and to Astar Winoto for Sp3 antiserum and plasmid DNA used in this study. This work was supported in part by National Cancer Institute, National Institutes of Health grant CA53248 and Faculty Research Award A-73970 from the American Cancer Society to J.M.H. J.M.H. is a member of the Pew Scholars Program in the Biomedical Sciences.

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*To whom correspondence should be addressed. Tel: +1 919 613 8617; Fax: +1 919 613 8604; Email: jmh@galactose.mc.duke.edu

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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				M. L. Spengler and M. G. Brattain Sumoylation Inhibits Cleavage of Sp1 N-terminal Negative Regulatory Domain and Inhibits Sp1-dependent Transcription J. Biol. Chem., March 3, 2006; 281(9): 5567 - 5574. [Abstract] [Full Text] [PDF]

				S. Das, S. V. Ward, R. S. Tacke, G. Suske, and C. E. Samuel Activation of the RNA-dependent Protein Kinase PKR Promoter in the Absence of Interferon Is Dependent Upon Sp Proteins J. Biol. Chem., February 10, 2006; 281(6): 3244 - 3253. [Abstract] [Full Text] [PDF]

				A. Lambiase, D. Merlo, C. Mollinari, P. Bonini, A. M. Rinaldi, M. D Amato, A. Micera, M. Coassin, P. Rama, S. Bonini, et al. Molecular basis for keratoconus: Lack of TrkA expression and its transcriptional repression by Sp3 PNAS, November 15, 2005; 102(46): 16795 - 16800. [Abstract] [Full Text] [PDF]

				R. R. Sharma, T. S. Ravikumar, D. Raimo, and W.-L. Yang Induction of p21WAF1 Expression Protects HT29 Colon Cancer Cells From Apoptosis Induced by Cryoinjury Ann. Surg. Oncol., September 1, 2005; 12(9): 743 - 752. [Abstract] [Full Text] [PDF]

				H. S. Choi, C. K. Hwang, C. S. Kim, K. Y. Song, P.-Y. Law, L.-N. Wei, and H. H. Loh Transcriptional Regulation of Mouse {micro} Opioid Receptor Gene: Sp3 Isoforms (M1, M2) Function as Repressors in Neuronal Cells to Regulate the {micro} Opioid Receptor Gene Mol. Pharmacol., May 1, 2005; 67(5): 1674 - 1683. [Abstract] [Full Text] [PDF]

				S. A. Irvine, P. Foka, S. A. Rogers, J. R. Mead, and D. P. Ramji A critical role for the Sp1-binding sites in the transforming growth factor-{beta}-mediated inhibition of lipoprotein lipase gene expression in macrophages Nucleic Acids Res., March 8, 2005; 33(5): 1423 - 1434. [Abstract] [Full Text] [PDF]

				G. Pages and J. Pouyssegur Transcriptional regulation of the Vascular Endothelial Growth Factor gene-a concert of activating factors Cardiovasc Res, February 15, 2005; 65(3): 564 - 573. [Abstract] [Full Text] [PDF]

				T. Hashimoto, T. Nakamura, H. Maegawa, Y. Nishio, K. Egawa, and A. Kashiwagi Regulation of ATP-sensitive Potassium Channel Subunit Kir6.2 Expression in Rat Intestinal Insulin-producing Progenitor Cells J. Biol. Chem., January 21, 2005; 280(3): 1893 - 1900. [Abstract] [Full Text] [PDF]

				G. Tsika, J. Ji, and R. Tsika Sp3 Proteins Negatively Regulate {beta} Myosin Heavy Chain Gene Expression during Skeletal Muscle Inactivity Mol. Cell. Biol., December 15, 2004; 24(24): 10777 - 10791. [Abstract] [Full Text] [PDF]

				Z. Ma, M. J. Chang, R. Shah, J. Adamski, X. Zhao, and E. N. Benveniste Brg-1 Is Required for Maximal Transcription of the Human Matrix Metalloproteinase-2 Gene J. Biol. Chem., October 29, 2004; 279(44): 46326 - 46334. [Abstract] [Full Text] [PDF]

				A. Sapetschnig, F. Koch, G. Rischitor, T. Mennenga, and G. Suske Complexity of Translationally Controlled Transcription Factor Sp3 Isoform Expression J. Biol. Chem., October 1, 2004; 279(40): 42095 - 42105. [Abstract] [Full Text] [PDF]

				T. Sato and K. Furukawa Transcriptional Regulation of the Human {beta}-1,4-Galactosyltransferase V Gene in Cancer Cells: ESSENTIAL ROLE OF TRANSCRIPTION FACTOR Sp1 J. Biol. Chem., September 17, 2004; 279(38): 39574 - 39583. [Abstract] [Full Text] [PDF]

				L. Pan, C. A. Jones, S. T. Glenn, and K. W. Gross Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1107 - F1115. [Abstract] [Full Text] [PDF]

				K. S. Moorefield, S. J. Fry, and J. M. Horowitz Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells J. Biol. Chem., April 2, 2004; 279(14): 13911 - 13924. [Abstract] [Full Text] [PDF]

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