Sp-1 Binds Promoter Elements That Are Regulated by Retinoblastoma and Regulate CTP:Phosphocholine Cytidylyltransferase-α Transcription
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Bibliographic record
Abstract
The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection 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 transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-α (CTα) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTα transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTα expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTα expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTα activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation. The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection 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 transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-α (CTα) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTα transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTα expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTα expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTα activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation. Phosphatidylcholine (PC) 3The abbreviations used are: PC, phosphatidylcholine; CT; CTP:phosphocholine cytidylyltransferase; FBS, fetal bovine serum; LUC, luciferase; Rb, retinoblastoma protein; RCE, retinoblastoma control element; RCP, retinoblastoma control protein; qPCR, quantitative PCR; CMV, cytomegalovirus; ChIP, chromatin immunoprecipitation. is the major phospholipid in mammalian cells and is a precursor for the synthesis of sphingomyelin and phosphatidylserine. PC biosynthesis occurs in all nucleated mammalian cells via the Kennedy (CDP-choline) pathway in which CTP:phosphocholine cytidylyltransferase (CT) catalyzes the regulated and rate-limiting step (1Vance D.E. Biochem. Cell Biol. 1990; 68: 1151-1165Crossref PubMed Scopus (147) Google Scholar, 2Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (240) Google Scholar, 3Lykidis A. Jackson P. Jackowski S. Biochemistry. 2001; 40: 494-503Crossref PubMed Scopus (41) Google Scholar). Two genes (Pcyta1 and Pcytb1, for CTα and CTβ, respectively) encode CT activity (4Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 5Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 6Tang W. Keesler G.A. Tabas I. J. Biol. Chem. 1997; 272: 13146-13151Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 7Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. AcaSci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar, 8Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar). CTα is ubiquitously expressed in nucleated cells (9Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and its expression is tightly regulated. CTα is also regulated post-translationally by reversible association with membrane lipids, which are required for its activity (10Tronchere H. Record M. Terce F. Chap H. Biochim. Biophys. Acta. 1994; 1212: 137-151Crossref PubMed Scopus (98) Google Scholar, 11Cornell R.B. Biochem. Soc. Trans. 1998; 26: 539-544Crossref PubMed Scopus (27) Google Scholar, 12Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar, 13Pelech S.L. Cook H.W. Paddon H.B. Vance D.E. Biochim. Biophys. Acta. 1984; 795: 433-440Crossref PubMed Scopus (77) Google Scholar, 14Pelech S.L. Pritchard P.H. Brindley D.N. Vance D.E. J. Biol. Chem. 1983; 258: 6782-6788Abstract Full Text PDF PubMed Google Scholar). At the level of gene expression, CTα mRNA has been shown to increase after growth factor stimulation (15Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar), during liver development (16Sesca E. Perletti G.P. Binasco V. Chiara M. Tessitore L. Biochem. Biophys. Res. Commun. 1996; 229: 158-162Crossref PubMed Scopus (29) Google Scholar), in proliferating liver tissue following partial hepatectomy (17Houweling M. Cui Z. Tessitore L. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar), and during the S phase of the cell cycle (18Golfman L.S. Bakovic M. Vance D.E. J. Biol. Chem. 2001; 276: 43688-43692Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We recently reported that the expression of CTα is activated in late G1-S phase by the action of Sp1 (19Banchio C. Schang L.M. Vance D.E. J. Biol. Chem. 2003; 278: 32457-32464Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and repressed in quiescent cells by the action of histone deacetylase (20Banchio C. Lingrell S. Vance D.E. J. Biol. Chem. 2006; 281: 10010-10015Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The retinoblastoma gene product (Rb) was the first tumor suppressor to be identified (21Friend S.H. Bernards R. Rogelj S. Weinberg R.A. Rapaport J.M. Albert D.M. Dryja T.P. Nature. 1986; 323: 643-646Crossref PubMed Scopus (2220) Google Scholar). Rb regulates the expression of several genes through cis-acting elements in a cell type-dependent manner through the retinoblastoma control element (RCE), which is present in some genes responsible for cell growth and differentiation (22Mulligan G. Jacks T. Trends Genet. 1998; 14: 223-229Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). This element can be positively or negatively regulated by Rb and by three nuclear proteins of 115, 95, and 80 kDa (retinoblastoma control proteins (RCPs)) that bind RCE in vitro (23Kim S.J. Onwuta U.S. Lee Y.I. Li R. Botchan M.R. Robbins P.D. Mol. Cell. Biol. 1992; 12: 2455-2463Crossref PubMed Scopus (224) Google Scholar, 24Robbins P.D. Horowitz J.M. Mulligan R.C. Nature. 1990; 346: 668-671Crossref PubMed Scopus (223) Google Scholar, 25Creagh S.C. Robbins J.M. Littlejohn R.G. Physical Rev. A. 1990; 42: 1907-1922Crossref PubMed Scopus (120) Google Scholar, 26Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth & Differ. 1992; 3: 597-608PubMed Google Scholar). The RCPs bind to the RCEs within the c-fos, c-myc, and transforming growth factor-β1 promoters (26Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth & Differ. 1992; 3: 597-608PubMed Google Scholar). RCPs were shown to be members of the Sp1 transcription factor family. The 115- and 95-kDa RCP proteins correspond to Sp1 and Sp3 transcription factors, respectively, and activate RCE-mediated transcription. In contrast, the 80-kDa RCP, which is produced by internal initiation of transcription from Sp1 mRNA, acts as a potent inhibitor of Sp1/Sp3-mediated transcription (26Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth & Differ. 1992; 3: 597-608PubMed Google Scholar, 27Udvadia A.J. Rogers K.T. Higgins P.D. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar, 28Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 29Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (198) Google Scholar). RCP-mediated transcription is controlled by Rb protein (23Kim S.J. Onwuta U.S. Lee Y.I. Li R. Botchan M.R. Robbins P.D. Mol. Cell. Biol. 1992; 12: 2455-2463Crossref PubMed Scopus (224) Google Scholar). It seems that Rb regulates RCP-dependent transcription by direct interaction with RCPs rather than by binding to the RCE DNA sequences. Through sequence analysis, we discovered two putative RCEs in the promoter of the mouse Pcyta 1 gene. We explored the nature of these putative RCEs and their possible role in CTα regulation in C3H10T1/2 mouse embryonic fibroblasts. Materials—The luciferase vector, pGL3-basic, which contains the cDNA for Photinus pyraris luciferase, and the dual-luciferase Reporter Assay System were obtained from Promega (Madison, WI). Lipofectamine and PLUS reagent, Dulbecco's modified Eagle's medium, Schneider's medium, fetal bovine serum, and fetal calf serum were from Invitrogen. Anti-Rb and anti-Sp1 antibodies were from Santa Cruz Bio
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it