TY - JOUR
T1 - Functional Role for Sp1 in the Transcriptional Amplification of a Cell Cycle Regulated Histone H4 Gene
AU - Bimbaunv, Mark J.
AU - Wright, Kenneth L.
AU - van Wijnen, Andre J.
AU - Ramsey-Ewing, Anna L.
AU - Bourke, Michael T.
AU - Last, Thomas J.
AU - Aziz, Farah
AU - Frenkel, Baruch
AU - Rao, Brinda R.
AU - Aronin, Neil
AU - Stein, Gary S.
AU - Stein, Janet L.
PY - 1995/6
Y1 - 1995/6
N2 - The promoter of the cell cycle regulated histone FO108 H4 gene is mediated by two in vivo protein/DNA interaction domains, sites I and II. We have shown previously that site II mediates the cell cycle controlled enhancement of H4 gene transcription at the Gl/S phase boundary. Here we show that site I, an element containing both G-rich and ATF-like consensus sequences, confers maximal levels of transcription in proliferating cells. By the combined application of gel shift assays with site-directed mutagenesis, DNase I footprinting, oligonucleotide competition, in vitro expression of recombinant proteins, and specific antibody supershift studies, we demonstrate that the proximal G-rich sequence within site I interacts with the transcription factor Sp1, while the distal portion of site I interacts with members of the ATF family of proteins, including ATF-1. In vitro transcription studies as well as expression assays of transiently and stably transfected genes in HeLa cells reveal that the deletion of site I causes a dramatic decrease in expression. Mutation of the Sp1 element, which abolishes Sp1 binding, results in a 6-10- fold reduction in reporter activity. In addition, overexpression of Sp1 in Sp1-deficient cells results in the dramatic activation of the histone promoter. In contrast, mutation of the asymmetric ATF binding site, located distally within site I, has a more limited effect upon expression. Interestingly, the contribution of the Sp1 site to maximal transcription was cell type dependent. Thus, we demonstrate that the Sp1 binding site of the site I histone H4 promoter in particular is critical for maximal expression in living cells and postulate that this site may act to amplify the cell cycle response.
AB - The promoter of the cell cycle regulated histone FO108 H4 gene is mediated by two in vivo protein/DNA interaction domains, sites I and II. We have shown previously that site II mediates the cell cycle controlled enhancement of H4 gene transcription at the Gl/S phase boundary. Here we show that site I, an element containing both G-rich and ATF-like consensus sequences, confers maximal levels of transcription in proliferating cells. By the combined application of gel shift assays with site-directed mutagenesis, DNase I footprinting, oligonucleotide competition, in vitro expression of recombinant proteins, and specific antibody supershift studies, we demonstrate that the proximal G-rich sequence within site I interacts with the transcription factor Sp1, while the distal portion of site I interacts with members of the ATF family of proteins, including ATF-1. In vitro transcription studies as well as expression assays of transiently and stably transfected genes in HeLa cells reveal that the deletion of site I causes a dramatic decrease in expression. Mutation of the Sp1 element, which abolishes Sp1 binding, results in a 6-10- fold reduction in reporter activity. In addition, overexpression of Sp1 in Sp1-deficient cells results in the dramatic activation of the histone promoter. In contrast, mutation of the asymmetric ATF binding site, located distally within site I, has a more limited effect upon expression. Interestingly, the contribution of the Sp1 site to maximal transcription was cell type dependent. Thus, we demonstrate that the Sp1 binding site of the site I histone H4 promoter in particular is critical for maximal expression in living cells and postulate that this site may act to amplify the cell cycle response.
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U2 - 10.1021/bi00023a011
DO - 10.1021/bi00023a011
M3 - Article
C2 - 7779811
AN - SCOPUS:0029076773
SN - 0006-2960
VL - 34
SP - 7648
EP - 7658
JO - Biochemistry
JF - Biochemistry
IS - 23
ER -