Ets-1 activates the DRA promoter in B cells.

The X box in promoters of class II major histocompatibility complex genes plays a crucial role in the B-cell-specific and gamma interferon-inducible expression of these genes. The sequence TTCC is located in the pyrimidine tract which extends 5' to and partially overlaps the X box of the DRA promoter. This sequence resembles the core binding site for the Ets family of DNA-binding proteins. In this study, we demonstrate that mutations within the pyrimidine tract which change the TTCC motif, but do not affect the binding of regulatory factor X to the X box, decrease the activity of the DRA promoter in B cells. Furthermore, using electrophoretic mobility shift assays and cotransfection experiments, we demonstrate that Ets-1, but not Ets-2 or PU.1, functionally interacts with the pyrimidine tract and activates the DRA promoter.     

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.     

Ets-1 and Ets-2 protooncogene expression in theca cells of the adult mouse ovary.

We have investigated the mRNA expression of the Ets-1 and Ets-2 genes in murine gonads and found expression in adult ovaries. In situ hybridization experiments show that the Ets genes are predominantly expressed in theca cells and cells of ovarian interstitium. By gel retardation experiments we detected DNA binding proteins in ovaries that specifically bind to the ETS motif, suggesting the expression of Ets or Ets-related proteins. Our results raise the possibility of Ets-2 involvement in ovarian pathology seen in patients with Down's syndrome.     

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.     

Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo

Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (–902 to –656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.