The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.     

One of two Ets-binding sites in the cytokeratin EndoA enhancer is essential for enhancer activity and binds to Ets-2 related proteins.

Expression of the mouse cytokeratin EndoA gene is restricted in endodermal and epithelial cells, and is regulated by an enhancer that is located 1 kilobase (kb) 3' downstream from the gene. The enhancer consists of six direct repeats, of which each contains two predicted Ets binding sites (EBS1 and EBS2) containing GGAA as a core. Mutation analysis showed that EBS1 is essential for the enhancer activity and additional effects of EBS2, suggesting that some Ets-related proteins bind and activate the enhancer through EBS1. We also showed that Ets-2 mRNA is expressed in PYS-2 cells and that Ets-2 protein produced by E. coli interacts with EBS1 but not with EBS2. Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells. Mutations that impair Ets-2 binding abolished the activity of the EndoA enhancer. The results obtained from the binding competition assay using an Ets-2 specific antibody, however, suggest that EBS1 binds to an Ets protein which is distinct from Ets-2. These data show that Ets-2 related protein binds and activates the EndoA enhancer in a sequence-specific fashion.     

Coordinate regulation of basal and cyclic 5'-adenosine monophosphate (cAMP)-activated expression of human chorionic gonadotropin-alpha by Ets-2 and cAMP-responsive element binding protein

Ets-2 controls the activities of many genes characteristically up-regulated in trophoblast. One apparent exception has been the gene for the human chorionic gonadotropin subunit alpha (hCGalpha). Here, we show that the hCGalpha gene contains two overlapping Ets binding sites adjacent to an activator protein-1-like site in its proximal promoter. Transactivation by Ets-2 is susceptible to truncation and mutation of these sites, which bind Ets-2 during in vitro mobility shift assays, as well as in vivo as determined by chromatin immunoprecipitation in choriocarcinoma cells. Knockdown of Ets-2 with short interfering RNA decreases both promoter activity and synthesis of hCGalpha. Ets-2 acts in combination with the protein kinase A (PKA) signal transduction pathway to activate the hCGalpha promoter expression. Mutation of the Ets-2 binding sites dramatically reduces up-regulation by PKA, whereas mutations within the two cAMP-responsive elements abolish responsiveness of the promoter to Ets-2. cAMP-responsive element binding protein (CREB) and Ets-2 form a complex that can be coimmunoprecipitated from choriocarcinoma cells, and association of CREB and Ets-2 is increased by activation of PKA. Regulation of hCGalpha subunit gene activity by cAMP involves the binding of CREB and Ets-2 to discrete elements in the promoter as well as a physical interaction between the two proteins. We propose that regulation of hCGalpha by Ets-2 and CREB enables coordinated expression of hCGalpha with its partner hCGbeta subunit.     

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.     

Mouse integrin alphav promoter is regulated by transcriptional factors Ets and Sp1 in melanoma cells

A 17-bp region between the -31 and -15 bp region of the mouse integrin alphav gene is known to be one of the cis-acting elements for promoter activity. Experimental binding of nuclear proteins to the -31/-15 region reveals that the -27/-16 region mediates the binding. The -27/-16 region, GGCTCCTCCTCC, has a TCCTCC motif, one of the Sp1 binding motifs. An anti-Sp1 IgG and an Sp1-binding oligonucleotide interfered with the binding of nuclear proteins to the -27/-16 oligonucleotide, demonstrating that Sp1 binds to the -27/-16 region. In addition to the -27/-16 region, two other regions, -108/-89 and -64/-44, were found to bind to nuclear proteins within the -108/+1 alphav promoter region. An oligonucleotide containing the Ets-binding consensus sequence of CAGGAAGT interfered with their binding, indicating that both regions have a functional Ets-binding site; which is ACGGAAGT from -106 to -99 bp and ACTTCCTC from -61 to -54 bp, as deduced from the sequence. Mutations in or deletions from any one of three cis-acting elements, the two Ets-binding sites or one Sp1-binding site, remarkably decreased the promoter activity detected in the -108/+1 region. Cotransfection of both Sp1 and Ets-1 cDNAs with the -108/+1 region into B16F10 cells increased the promoter activity 2.9-fold. These results demonstrate that Sp1 and Ets cooperate to activate the -108/+1-alphav promoter region.