Functional role for Sp1 in the transcriptional amplification of a cell cycle regulated histone H4 gene.
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 G1/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.
Birnbaum
M J
MJ
Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655, USA.
Wright
K L
KL
van Wijnen
A J
AJ
Ramsey-Ewing
A L
AL
Bourke
M T
MT
Last
T J
TJ
Aziz
F
F
Frenkel
B
B
Rao
B R
BR
Aronin
N
N
D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006657:Histones / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D006657:Histones; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor