DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo.

E2F is a heterodimeric transcription factor that regulates the expression of genes at the G1/S boundary and is composed of two related but distinct families of proteins, E2F and DP. E2F/DP heterodimers form complexes with the retinoblastoma (Rb) protein, the Rb-related proteins p107 and p130, and cyclins/cdks in a cell cycle-dependent fashion in vivo. E2F is encoded by at least five closely related genes, E2F-1 through -5. Here we report studies of DP-2, the second member of the DP family of genes. Our results indicate that (i) DP-2 encodes at least five distinct mRNAs, (ii) a site of alternative splicing occurs within the 5' untranslated region of DP-2 mRNA, (iii) at least three DP-2-related proteins (of 55, 48, and 43 kDa) are expressed in vivo, (iv) each of these proteins is phosphorylated, and (v) one DP-2 protein (43 kDa) carries a truncated amino terminus. Our data also strongly suggest that the 55-kDa DP-2-related protein is a novel DP-2 isoform that results from alternative splicing. Thus, we conclude that DP-2 encodes a set of structurally, and perhaps functionally, distinct proteins in vivo.

Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain.

Sp1 is a well characterized and ubiquitously expressed transcription factor that regulates the constitutive and induced expression of a variety of mammalian genes. It is unclear whether Sp1 activity is regulated in vivo; the mechanism by which Sp1 interacts with the basal transcription complex has not been firmly established. We report the identification of a ubiquitously expressed and evolutionarily conserved nuclear protein, p74, that specifically binds Sp1 in vivo and in vitro. p74 interacts with several portions of the Sp1 trans-activation domain in vitro, and we correlate the binding of p74 to the amino-terminal serine/threonine-rich subdomain of Sp1 with the inhibition of Sp1-mediated transcription in vivo.

Sp-1 binds promoter elements regulated by the RB protein and Sp-1-mediated transcription is stimulated by RB coexpression.

The retinoblastoma (RB) protein is implicated in transcriptional regulation of at least five cellular genes, including c-fos, c-myc, and transforming growth factor beta 1. Cotransfection of RB and truncated promoter constructs has defined a discrete element (retinoblastoma control element; RCE) within the promoters of each of these genes as being necessary for RB-mediated transcription control. Previously, we have shown that RCEs form protein-DNA complexes in vitro with three heretofore unidentified nuclear proteins and mutation of their DNA-binding site within the c-fos RCE results in an abrogation of RCE-dependent transcription in vivo. Here, we demonstrate that one of the nuclear proteins that binds the c-fos, c-myc, and transforming growth factor beta 1 RCEs in vitro is Sp-1 and that Sp-1 stimulates RCE-dependent transcription in vivo. Moreover, we show that Sp-1-mediated transcription is stimulated by the transient coexpression of RB protein. We conclude from these observations that RB may regulate transcription in part by virtue of its ability to functionally interact with Sp-1.

A common set of nuclear factors bind to promoter elements regulated by the retinoblastoma protein.

A 30-base pair element within the c-fos promoter, termed the RCE (retinoblastoma control element), has previously been shown to be the target of transcriptional regulation by the product of the retinoblastoma (Rb) gene. We have identified three nuclear proteins [retinoblastoma control proteins (RCPs)] that complex with this promoter element in vitro. The Rb gene does not appear to encode the RCPs as the expression of Rb in vivo does not correlate with RCE-RCP complex formation in vitro. A single binding site for the RCPs within the c-fos RCE was identified, and the nucleotides required for protein-DNA complex formation were defined. Similar sequences are found in the promoters of two additional genes that are regulated by Rb (c-myc and TGF-beta 1), and binding assays demonstrate that the RCPs also interact with these elements. Linkage of the c-fos RCE to the herpes simplex virus thymidine kinase promoter led to a 4-fold stimulation of expression in transient transfection assays. Mutations within the RCP binding site that abrogate stable interaction of the RCPs with the RCE in vitro block RCE transcriptional activity in vivo. Our results suggest a role for the RCPs in RCE-dependent transcription and the regulation of transcription by the Rb protein.

Expression of the TGF alpha integral membrane precursor induces transformation of NRK cells.

TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.

Characterization of the rat transforming growth factor alpha gene and identification of promoter sequences.

We have determined the complete nucleotide sequence of rat transforming growth factor alpha (TGF alpha) mRNA and characterized the six exons that encode this transcript. These six exons span approximately 85 kilobases of genomic DNA, with exons 1 to 3 separated by particularly large introns. What had previously been thought to represent a species-specific difference in the size of the TGF alpha precursor (proTGF alpha) is now shown to be due to microheterogeneity in the splicing of exons 2 and 3. This results from a tandem duplication of the acceptor CAG and gives rise to two alternate forms (159 and 160 amino acids) of the integral membrane precursor. Exon 6, which encodes the 3' untranslated region of TGF alpha mRNA, also encodes, on the opposite strand, a small (approximately 200-nucleotide) transcript whose sequence predicts an open reading frame of 51 amino acids. Expression of this latter transcript does not appear to be coregulated with that of TGF alpha mRNA. Primer extension and S1 nuclease analyses of authentic TGF alpha transcripts revealed two major and multiple minor 5' ends which span more than 200 base pairs of DNA in a G + C-rich region that lacks canonical CCAAT or TATA sequences. The 5' ends of six independently derived cDNAs localized to five different sites in this same region. Restriction fragments that overlap these transcription start sites and extend approximately 300 base pairs in the 5' direction faithfully promote transcription in vitro with HeLa cell nuclear extracts. In addition, they direct the expression of the bacterial chloramphenicol acetyltransferase gene in transient-transfection assays.