Characterization of self-regulatory mechanisms and internal dynamics of ETS transcription factor PU.1

The ETS family of transcription factors bind to site-specific DNA via DNA-binding domains called the ETS domains. The ETS domains are structurally homologous but divergent in primary sequences. PU.1 is an essential transcription factor and its biological activity is primarily controlled by up- and down-regulation of its expression. Aside from down-regulated expression, only a few inhibitory mechanisms are known for PU.1. The most understood one involves PU.1 forming a heterodimer with other protein partners, such as GATA-1. However, unlike auto-inhibited ETS-family members whose activity is regulated by autoinhibitory elements that reduce the net affinity of binding to specific DNA, PU.1 has no such regulatory mechanism at the protein-DNA level. We report here that PU.1, unlike its auto-inhibited paralog Ets-1, forms a 2:1 complex with site-specific DNA (\u3e10 bp) in a negatively cooperative manner. We also detected potential interface (193DKDK196) of the PU.1 dimer by using heteronuclear single quantum correlation (HSQC) NMR. Self-titration of PU.1 is a negative feedback mechanism at the protein-DNA level. Following these findings, our group found the presence of the IDRs flanking the ETS domain does not change the DNA binding modes of the PU.1 ETS domain, yet the PEST domain modifies DNA recognition by the ETS domain through changing DNA binding affinities. We successfully assigned ~90% or more backbone amide resonances in the 1H-15N HSQC spectra of hPU.1 constructs with and without IDRs, in the absence and presence (1:1 complex) of DNA. Using the fully assigned HSQC spectra, we studied fast (ps to ns) time scale internal dynamics of PU.1 protein. Spin relaxation rates and heteronuclear 1H{15N}-NOE were acquired for the hPU.1 proteins with and without DNA by NMR. We demonstrated that the PEST domain remains disordered but becomes more dynamic upon specific DNA binding. In terms of DNA recognition, the presence of the PEST domain increases the affinity of 1:1 complex of the ETS domain with cognate DNA, without perturbing the structure or changing the fast time scale backbone motions of the ETS domain