Identification of ETS domain proteins in murine T lymphocytes that interact with the Moloney murine leukemia virus enhancer.

Abstract

The enhancer of Moloney murine leukemia virus (Mo-MuLV) contains an array of transcriptional control elements that direct viral gene expression in diverse cell types. The murine transcription factor Ets-1 was shown to bind to the LVb and LVc elements of the enhancer by DNase I protection and methylation interference assays. Enhancers containing disrupted Ets-1 binding sites were tested in transient expression assays in the murine T-cell line EIA.El; alterations in the LVb element affected constitutive enhancer activity, while mutation of either the LVb or LVc element disrupted phorbol ester-induced enhancer activity. Members of the ets gene family of proteins display similar DNA-binding properties; therefore, we speculated that ets proteins other than Ets-1 also might bind these elements. Crude nuclear extracts of EL4.E1 cells were assayed to identify the protein(s) that potentially functions at the LVb element. The predominant binding activity was not Ets-1 but rather two independent DNA-protein complexes that comigrated in mobility shift assays. UV cross-linking and denaturing gel electrophoresis sized the two DNA-binding species, which we denoted p55 and plOO. Immunoprecipitation combined with UV cross-linking identified p55 as the alpha subunit of GA-binding protein. The DNA-binding properties of plOO and several ets proteins were compared. Similarities suggested that plOO is also an ETS domain protein, possibly Elf-1. This strategy could be used to identify other ETS domain proteins in crude nuclear extracts. These findings suggest multiple ETS domain proteins could regulate gene expression of Mo-MuLV.Moloney murine leukemia virus (Mo-MuLV) provides a useful model for the study of eukaryotic transcriptional machinery. The transcriptional control elements in the long terminal repeat (LTR) include a TATA box and an extensive array of positive-acting elements arranged as a proximal promoter and a more distal enhancer (13,25). Many cellular proteins that bind these activator elements have been identified (7,14,17,38,50). Transcription factors that regulate the Mo-MuLV enhancer in T cells are of particular interest since Mo-MuLV causes T-cell lymphomas and leukemias in mice. Regulatory sequences within the enhancer have been identified as determinants for both thymotropism and T-cell transformation (5,8,11,28,39,40). However, the T-cell specificity of virus-induced disease does not preclude the function of the Mo-MuLV LTR in many other cell types; LTR-driven transcription as well as viral replication have been observed in numerous tissue types and mammalian cell lines in culture (56). These data suggest that Mo-MuLV can use different combinations of transcription factors, depending on the cell type. Insights bearing on this issue come from the discovery that members of the ets family of eukaryotic transcription factors bind key regulatory elements in the Moloney LTR.The ets gene family of proteins includes transcription factors that display conservation within an 85-residue DNA-binding domain, termed the ETS dom...

Sequence Discrimination by DNA-binding Domain of ETS Family Transcription Factor PU.1 Is Linked to Specific Hydration of Protein-DNA Interface

“…Specific hydration might therefore evolve as compensatory mechanism to replace some of the "missing" anhydrous contacts. The involvement of specifically bound water at the backbone contacts by PU.1 is also consistent with the substantially smaller DNA curvature (8°) compared with Ets-1 (27°) and GABP␣ (18°) despite sharing identical phosphate contacts (25). Finally, high affinity binding for the minimal ETS domain of Ets-1 exhibits dissociation constants at 10 Ϫ12 M under physiological conditions (43).…”

“…Specifically, the TTCC strand shows a strongly hypersensitive band at N 1 (5Ј-TpTpCpCpN 1 2pN 2 pT-3Ј) just 3Ј to the core consensus. This hypersensitivity is a universal hallmark of sequencespecific ETS-DNA complexes (22)(23)(24)(25)(26). It is attributed to a widening of the minor groove by the recognition helix (h3) at the major groove of the core consensus.…”

“…Hypersensitivity to DNase I, a minor groove probe, is an invariant feature of sequence-specific ETS-DNA complexes (22)(23)(24)(25)(26). Because minor groove width is defined by residues i and i ϩ 2 in the complementary strand (38), the [Ϫ] flanking bases in the TTCC strand (5Ј-GCT-3Ј in [Ϫ]GC, 5Ј-TTT-3Ј in [ϩ]TG) are directly involved in defining the minor groove width of the core consensus.…”

Oestrogen and progesterone inhibit the stimulated production of endothelin-1

“…Although there may be auxiliary positive and negative regulatory elements between nucleotides k143 and k43, these data suggests that the element(s) primarily necessary for serum-induced ET-1 transcription lies within the first 43 bp of 5h-flanking promoter sequence. In scanning this sequence, a few possible binding sites for known transcription factors were found, including LVc, TCF-1 and GMCSF-CS sites [38][39][40][41][42][43]. However, these factors have only been found in T-and Blymphocytes.…”

Defective Production of Functional 98-kDa Form of Elf-1 Is Responsible for the Decreased Expression of TCR ζ-Chain in Patients with Systemic Lupus Erythematosus

“…Previous studies have indicated that promoter-defined Elf-1 sites from different genes bind preferentially different forms of Elf-1. The human IL-2 promoterdefined Elf-1 site binds all forms of Elf-1 (14), whereas the enhancer of the Moloney murine leukemia virus binds the 98-kDa form of Elf-1 (15).…”