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. 2003 Dec;23(23):8773-85.
doi: 10.1128/MCB.23.23.8773-8785.2003.

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription

Li-Chung Hsu  1 , Shu Liu, Ferishteh Abedinpour, Robert D Beech, Jill M Lahti, Vincent J Kidd, Jeffrey A Greenspan, Cho-Yau Yeung
Affiliations

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription

Li-Chung Hsu et al. Mol Cell Biol. 2003 Dec.

Abstract

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

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Figures

FIG. 1.
FIG. 1.
mGPBP sequence and sequence homology. (A) Complete cDNA sequence corresponding to the 3.0-kb murine GPBP mRNA, including the deduced amino acid sequence encoded by the ORF (GenBank accession number AY382529). Sequence homology searches revealed small regional sequence homology on the amino acid sequence level with several known proteins as shown in panel B. Solid box, ORF; open boxes, regions homologous to the yeast TFIIFα subunit (yTFIIFα), yABF-1, murine SSRP1 (mHMG-1 like), and helicases.
FIG. 2.
FIG. 2.
Northern blot analyses to examine the mGPBP mRNA species and tissue distribution. Approximately 3 μg of poly(A+) RNA derived from the tissues indicated was loaded per lane. (A) The blot was first probed with an mGPBP-specific probe (top) and then stripped and reprobed with a β-actin probe (bottom). (B) The blot was probed with a 2.0-kb probe derived from the 5′ terminus of the mGPBP cDNA (left), stripped, and reprobed with a 350-bp probe derived from the 3′ terminus of the mGPBP cDNA corresponding to the 3.5-kb mGPBP mRNA (right). The 5′ probe hybridized with both mRNA species, whereas the 3′ probe hybridized with only the 3.5-kb mRNA.
FIG. 3.
FIG. 3.
Mammalian GPBP is approximately 66 kDa in size and is ubiquitously expressed. (A) Electrophoretically separated proteins in SDS-polyacrylamide gels stained with Coomassie blue. Bacterial lysate derived from a BL21 strain carrying the PET-mGPBP expression plasmid was analyzed either without (−) or with (+) IPTG induction. Lane 4, purified recombinant mGPBP; lanes 1 and 5, molecular mass markers; lanes 7 and 8, Western blot analysis of the samples used in lanes 3 and 4, respectively, using antiserum against mGPBP as the probe; lane 6, analysis of control BL21 cell (without the PET-mGPBP expression plasmid) lysate. (B) Western blot analyses of cell lysates derived from human HeLa cells (lane 1), mouse Cl-1D cells (lane 2), human JEG-3 cells (lane 3), human VA-2 cells (lane 4), mouse M2-10B4 cells (lane 5), and human 293 cells (lane 6) using the anti-mGPBP antiserum as the probe.
FIG. 4.
FIG. 4.
The purified recombinant mGPBP can bind specifically to the mouse Ada gene's G+C-rich promoter in EMSA. (A) The DNA probe 4C′ is four copies of the MSPE C′ that were end ligated. Purified recombinant mGPBP (rmGPBP) bound specifically to the probe and caused a shift in probe electrophoretic mobility from the free-probe location (lane 1) to the bound-probe location (lane 2). This binding can be specifically competed out by adding 35-fold (lane 3) and 175-fold (lane 4) excess unlabeled probes but cannot be competed out by adding similar amounts of unlabeled E2F binding motifs (lanes 5 and 6) or a 200-bp plasmid sequence (lanes 7 and 8). (B) A single copy of the fragment C′ in the context of the labeled 236-bp mouse Ada gene promoter can also bind to and be electrophoretically retarded by the purified rmGPBP (lanes 1 and 2). This binding can be competed out by excess unlabeled probe (lanes 3 and 4) or the 4C′ probe used in panel A (lanes 5 and 6). This binding is again not competed out by unlabeled E2F binding sequences (lanes 7 and 8) or the 200-bp plasmid sequences (lanes 9 and 10).
FIG. 5.
FIG. 5.
Both human and transfected mGPBP are localized in the nucleus. Human HeLa cells were stained with an antibody against mGPBP (N-80) (A, top) or an antibody from preimmune serum (B, top). The location of the nucleus was determined by counterstaining the cells with DAPI (A and B, bottom). The transfected HA-tagged mGPBP was localized with the αN-80 (C, top) and an anti-HA (C, bottom) antibodies.
FIG. 6.
FIG. 6.
The mGPBP can form complexes with multiple transcription initiation complex assembly specific factors. (A) Mouse Cl-1D cell nuclear extract proteins were allowed to bind to bead-immobilized GST-mGPBP fusion proteins. The various proteins, including the starting nuclear extract (input), proteins that bind to bead-immobilized GST or GST-mGPBP fusion proteins (lanes “bound to”), and proteins that did not bind to bead immobilized GST or GST-mGPBP fusion proteins (lanes “unbound supernatant”) were analyzed by Western blotting. The antibodies used to probe these blots were raised against TBP, the C-terminal domain of RNA polymerase II (RNA pol II CTD), TFIIB, TFIIF RAP30 subunit, P300/CBP, and the negative control, the nuclear envelope protein nucleoporin p62. The estimated molecular masses of proteins in each band based on electrophoretic mobility in comparison to protein size markers are on the right. (B) In vivo binding of mGPBP was determined by coimmunoprecipitation analyses of cell lysates. Western blot analyses showed Cl-1D cell lysate proteins after the cells were transfected with expression vectors that express either the HA tag (HA) alone or the HA tag fused to mGPBP (HA-mGPBP), without immunoprecipitation (input lysate) or after immunoprecipitation with antibodies against the HA tag (IP αHA Ab). The antibodies raised against TBP, RNA pol II CTD, TFIIB, CBP, and the HA tag were used as probes as indicated in each blot. The estimated molecular masses of proteins in each band based on electrophoretic mobility in comparison to protein size markers are shown. The migration location of the HA-mGPBP fusion protein is also shown on the right.
FIG. 7.
FIG. 7.
mGPBP is specifically required for transcription initiated at the mouse Ada gene's G+C-rich promoter. Cotransfection of a mouse Ada gene promoter-controlled luciferase reporter gene construct together with increasing amounts of a mGPBP expression vector into either mouse Cl-1D cells or human 293 cells resulted in a linear increase in reporter gene activity (A). In vitro transcription assays performed by coincubation of a supercoiled mouse Ada gene promoter-controlled G-less cassette reporter gene with human HeLa cell nuclear extracts showed that reporter transcript production was unaltered by the addition of increasing amounts of preimmune serum-derived antibodies (B, lanes 1 to 3). In contrast, increasing amounts of anti-mGPBP antibodies caused a gradual decrease in reporter transcript production (B, lanes 4 to 9). This immunosuppression effect can be reversed by the addition of purified recombinant mGPBP (rmGPBP; B, lanes 10 and 11). Transcription of the G-less cassette reporter gene under the control of the TATA box-dependent adenovirus major late promoter was not affected by the addition of anti-mGPBP antibodies (B, lanes 12 to 15). The assay results were repeated at least three times. (C) Normalized quantitation of the transcription assay results (using a phosphorimager), together with standard deviations of multiple repetitions. The statistical significance (P) in the difference of transcription levels in the absence and presence of anti-mGPBP antibodies (*) was found by a paired Student t test to be <0.00042 (n = 8), and P for the difference of transcription levels in the presence of the anti-mGPBP antibodies with and without additional recombinant mGPBP (†) was found by a paired Student t test to be <0.024 (n = 4).
FIG. 8.
FIG. 8.
The TATA box containing the Topo IIα gene's G+C-rich promoter binds specifically to mGPBP but is only partially dependent on its presence for promoter function. (A) The human Topo IIα gene promoter shows no obvious sequence homology to the Ada gene's MSPE. The sequences displaying an imperfect dyad symmetry flanking the major transcription initiation site (arrow) are underlined. The consensus TATA element in the Topo IIα gene promoter is boxed. (B) Electrophoretic mobility of the 32P-labeled Topo IIα gene promoter probe (lane 1, arrow) was retarded (bound probe) by the presence of purified recombinant mGPBP (lane 2). This retardation of the probe can be reversed by competition with excess unlabeled probe (lanes 3 and 4) or linked quadruple copies of the MSPE derived from the Ada gene promoter (lanes 7 and 8), but not by excess copies of a 200-bp plasmid sequence that shows no secondary conformational changes under negative superhelicity (lanes 5 and 6). (C) In vitro transcription of supercoiled reporter genes driven by the Topo IIα gene promoter with HeLa nuclear extract was partially suppressed by the presence of anti-GPBP antibodies but was unaffected by the presence of preimmune antibodies. The suppressive effect of the anti-GPBP antibodies can be fully reversed by the addition of 20 ng of purified recombinant mGPBP to the reaction mixture. The statistical significance (P) for the difference of transcription levels in the absence and presence of anti-mGPBP antibodies (*) was found by a paired Student t test to be <0.000003 (n = 5), and P for the difference of transcription levels in the presence of the anti-mGPBP antibodies with and without additional recombinant mGPBP (†) was found by a paired Student t test to be <0.028 (n = 5).
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