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

Abstract

Background: PU.1 recognizes a large number of binding sites with differing affinities. Results: The role of hydration in sequence-specific binding by the ETS domain of PU.1 was determined. Conclusion: Sequence discrimination is linked to the uptake of specifically bound waters at the protein-DNA interface. Significance: The sequence specificity of PU.1 ETS is directly related to the transactivational activity and frequency of in vivo binding sites for the full-length protein.

Mechanistic Heterogeneity in Site Recognition by the Structurally Homologous DNA-binding Domains of the ETS Family Transcription Factors Ets-1 and PU.1

“…Molecular Cloning-The DNA sequence encoding the ETS domain of murine PU.1 (residues 167-272, termed ⌬N167) was cloned into pQE60 as previously described (18). The minimal ETS domain of human Ets-1 (residues 311-440, termed ⌬N311) was amplified by PCR from full-length Ets-1 (GenBank TM accession number AY888522.1) into the NcoI/BamHI sites of pET28b.…”

“…Protein Expression and Purification-The recombinant ETS domain of PU.1 was overexpressed in Escherichia coli and purified as previously described (18). The Ets-1 constructs were handled similarly.…”

“…Recent studies on PU.1 have revealed an essential role for preferential hydration in sequence discrimination by PU.1 (18).…”

Signatures of DNA target selectivity by ETS transcription factors

“…62 We have found through analysis of microarray data that PU.1 target genes are disproportionately represented in osmotically sensitive (NFAT5-dependent) genes in primary murine macrophages. 61 Significantly, other transcription factors that are co-expressed with PU.1, such as the interferon regulatory factors, NFkB2, and Stat proteins, show no such overlap. While PU.1 may interact with NFAT5 by direct contact or via post-translational modification, no such interactions are currently known.…”

“…Specifically, we have observed that the different disposition of hydration water directly impacts on the mechanisms of DNA target recognition as manifest in their kinetics of association and dissociation (Table 1). 61 Under normo-osmotic conditions, PU.1 engages sequence-specific target sites about »100 more slowly than Ets-1, but once formed, the complex is correspondingly more persistent than Ets-1. The starkly different kinetic profiles establish that interfacial hydration defines different mechanisms of target recognition by the two ETS homologs.…”