Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro.

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

Oncogenic Ras can induce transcriptional activation through a variety of promoter elements, including tandem c-Ets-2 binding sites.

Oncogenic Ras activates the transcription of a variety of viral and cellular genes through promoter elements consisting of two closely linked binding sites for transcription factors from several distinct families. To better understand what constitutes a promoter oncogene response element (ORE), various transcription factor binding site configurations were inserted into a reporter gene, and transactivation by oncogenic Ras was measured by cotransfection assays in NIH3T3 cells. We show that a single copy of two closely linked binding sites for either AP-1, Ets, NF-kappa B, or single closely linked Ets and AP-1 binding sites, are sufficient to confer at least 10-fold transactivation by similar amounts of oncogenic Ras. Single binding sites for these factors, or several other pairings of binding sites, are not sufficient to confer Ras responsiveness. The effect of altered ORE binding site spacing and orientation was systematically analysed, and limited flexibility was observed. The novel observation that two adjacent c-Ets-2 binding sites are sufficient to act as an ORE, indicates that Ets family proteins are a target of the Ras pathway distinct from AP-1. This ORE also mediates equivalent transactivation by c-Ets-2, and mutant OREs show a parallel decrease in Ras and c-Ets-2 responsiveness. Together, these data help to define transcriptional targets of the Ras signal transduction pathway.

Cooperative DNA binding of the highly conserved human Hox 2.1 homeodomain gene product.

The human homeobox-containing gene Hox 2.1 (hHox 2.1) and its murine cognate mHox 2.1 are part of evolutionarily conserved gene clusters, encode an identical Antennapedia-type homeodomain, and are expressed in a similar pattern in the developing embryo. We have isolated cDNA clones of hHox 2.1 and found that the human/murine Hox 2.1 gene structure is strikingly conserved, with only 3 out of 269 amino acid differences in the entire predicted protein sequence. We show that purified hHox 2.1 protein is a sequence-specific DNA binding protein capable of binding to a variety of DNA sequences, including multiple sites in the promoter of the hHox 2.1 gene. In a footprint titration assay, the apparent affinity of the hHox 2.1 protein for a consensus binding site (LP) increases when the site is present in tandem copies. Quantitative footprint challenge experiments revealed that the in vitro half-life of the protein-DNA complex is less than 30 s for a single LP binding site (kd greater than 1.4 min-1), but 126 min for proteins bound to two tandem LP binding sites (kd = 5.5 x 10(-3) min-1). A domain distinct from the homeodomain is necessary for cooperative DNA binding, because a 61-amino-acid peptide containing only the Hox 2.1 homeodomain can specifically bind to LP sites, but exhibits no cooperativity. A different full-length human homeodomain protein, hHox 1.3, was also found to show cooperative DNA binding quantitatively similar to hHox 2.1. Therefore, cooperative DNA binding to adjacent sites may be a crucial component in the overall affinity of mammalian Antennapedia-type homeodomain proteins for their DNA target sites.

Elevated expression of Ets2 or distinct portions of Ets2 can reverse Ras-mediated cellular transformation.

Ets transcription factors are important downstream targets of oncogenic Ras. The transcriptional activity of several Ets family members is regulated by Ras, and interfering with Ets-dependent transcription by expression of just the Ets2 DNA binding domain can inhibit or reverse Ras-mediated cellular transformation. To better understand the role of Ets proteins in Ras transformation, we have now analyzed the effects of stably expressing a variety of Ets2 constructs in Ras-transformed NIH3T3 (DT) cells. Expression of only the Ets2 transactivation domains, which also inhibits Ras or Neu/ErbB-2-mediated activation of Ets-dependent transcription, strongly inhibited anchorage-independent growth, but did not revert the transformed DT cell morphology. Unexpectedly, high expression of full-length Ets2, a transcriptional activator, broadly reversed the transformed properties of DT cells, including anchorage-independent growth, transformed morphology, and tumorigenicity, but did not impair attached cell growth. Increasing full-length Ets2 transcriptional activity by fusing it to the VP16 transactivation domain enhanced its ability to reverse DT cell transformation. Mutational analysis revealed that the mitogen-activated protein kinase phosphorylation site required for Ras-mediated activation, Ets2(T72), was not essential for Ets2 reversion activity. The distinct reversion activities of the highly expressed Ets2 transactivation domains or full-length Ets2, along with the specific reversion activity by Ets2 constructs that either inhibit or activate Ets-dependent transcription, suggests multiple roles for Ets factors in cellular transformation. These results indicate that several distinct approaches for modulating Ets activity may be useful for intervention in human cancers.

Oncogenic Neu/ErbB-2 increases ets, AP-1, and NF-kappaB-dependent gene expression, and inhibiting ets activation blocks Neu-mediated cellular transformation.

Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.