Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.     

NF-kappaB site interacts with Sp factors and up-regulates the NR1 promoter during neuronal differentiation

The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.     

Targeting of Sp1 to a non-Sp1 site in the human neurofilament (H) promoter via an intermediary DNA-binding protein.

The human neurofilament (H) promoter contains multiple binding sites for nuclear proteins including a Proximal (Prox) site centered around the sequence GGTTGGACC and an adjacent pyrimidine (Pyr) tract site centered around the sequence CCCTCCTCCCC. Surprisingly binding to a probe containing the Prox/Pyr region of the NF(H) promoter was competed in gel shifts by an oligonucleotide containing only an Sp1 binding site (GGGGCGGGG). Supershift assays with a polyclonal anti-Sp1 antisera confirmed that Sp1 was part of the complex formed with the Prox/Pyr probe. However neither bacterially expressed Sp1 516C or vaccinia virus expressed full-length Sp1 778C bound to the Prox or Pyr sequences in DNase I footprints or gel shift assays. Gel shift competitions and supershift assays with probes containing either Prox or Pyr tract sites alone demonstrated targeting of Sp1 to the Prox binding site and identified a non-Sp1 containing complex which contains a Prox binding protein. Adding exogenous Sp1 to a HeLa nuclear extract enhanced the Sp1-containing complex but had no effect on the Prox complex. These studies show that Sp1 can be targeted to a non-Sp1 site in the human NF(H) promoter through protein/protein interactions with a distinct sequence specific DNA-binding protein.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.