Sp3 Represses Gene Expression via the Titration of Promoter-specific Transcription Factors

Abstract

We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAF II s) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAF II 70 and TAF II 40, were identified. We conclude that Sp3-mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors.Sp1 is the founding member of a family of five transcription factors, Sp1-5, that govern the expression of a wide variety of mammalian genes (for review, see Ref. 1). Sp1 encodes a ubiquitously expressed nuclear phosphoprotein that has been divided into five sub-domains based upon their respective functions (2, 3). The Sp1 trans-activation domain is composed of three sub-domains termed A-C, each of which is capable of stimulating transcription if tethered to DNA via a DNA-binding domain. Sub-domains A and B are composed by serine-and threonine-rich regions as well as glutamine-rich regions. The glutamine-rich portions of A and B are believed to be required for trans-activation, whereas the function(s) of the serine/threonine-rich sub-regions is(are) less well understood. Domain C carries a number of charged amino acids and weakly stimulates transcription in the absence of domains A or B. Carboxylterminal to the domain C is a region featuring three Cys 2 -His 2 zinc "fingers" required for sequence-specific DNA binding to GC-rich promoter elements. A carboxyl-terminal domain, termed D, facilitates protein multimerization and is essential for synergistic trans-activation of promoters with multiple Spbinding sites. Sp1 associates with a large number of transcription-associated proteins, including components of the basal transcription complex (e.g. hTAF II 130/dTAF II 110 and hTAF II 55; Refs. 4 -7), sequence-specific DNA-binding proteins (e.g. E2F, YY1, p53, and AP-2; Refs. 8 -13), and transcriptional regulators (e.g. p107, HDAC-1, and VHL-1; Refs. 14 -16). As might be expected given the variety of proteins with which it interacts, protein-binding sites have been identified th...

Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells

“…Expression and Reporter Constructs-The construction and functional characterization of pPacSp1/flu, pPacSp3/flu, and pCMV4-Sp1/ flu have previously been described (23)(24)(25)(26). An Sp2 expression construct, pPacSp2/flu, carrying a 10-amino acid epitope from influenza hemagglutinin at the Sp2 carboxyl-terminus was prepared following a PCR that included a human Sp2 cDNA (a generous gift of Dr. Astar Winoto, University of California, Berkeley, CA) as template, Deep Vent polymerase (New England Biolabs, Inc., Beverly, MA), and the following primers: 5Ј-GGGGGATCCGCCACCATGGCGGCCGCCGTAATGA-GC-3Ј and 5Ј-GGGGGATCCCTAGCTAGCGTAATCTGGAACATCGTA-TGGGTACAAGTTCTTCGTGACCAG-3Ј.…”

“…Sf9 and SL2 cells were cultured as previously described (23)(24)(25). HTLA230 cells were a generous gift of Dr. Emil Bogenmann (Children's Hospital of Los Angeles, Los Angeles, CA).…”

“…Volumes of infected cell extracts that led to half-maximal binding of this probe were then employed in assays performed in triplicate with Sp-binding sites derived from the DHFR, p21, and MDR-1 genes and quantified in situ. GST fusion proteins were induced, purified, quantified, and utilized as previously described (24,25,27,33,34).…”

“…Total IgY was collected from eggs prior to and following four successive immunizations with immunogen (Aves Labs, Inc., Tigard, OR). Whole cell or nuclear extracts were resolved on denaturing polyacrylamide gels and transferred to nitrocellulose using a semidry transfer apparatus, and antigen-antibody complexes were detected as previously described (24,25,27,33,34).…”

“…SL2 cells are a convenient milieu for such studies, since they lack transcription factors closely related to Sp family members although they support Sp-mediated transcription. A full-length, human Sp2 cDNA was subcloned in an insect cell expression vector (pPac), and this construct or analogous constructions carrying Sp1 (pPacSp1/flu) or Sp3 (pPacSp3/flu) cDNAs were transiently co-transfected with a DHFR-luciferase reporter gene (DHFR-Lux) (25). Four Sp-regulated elements (termed DHFR1-DHFR4) have previously been identified and characterized within the 170-base pair portion of the DHFR promoter carried by DHFR-Lux.…”