STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

Abstract

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design.

Figure 1.

Analysis of the binding specificity of human TFs STAT1 and Myc/Max across DNA sequences containing cognate, mutant or no consensus binding sites in TIRF-PBM. (a) Equilibrium binding fluorescence intensities of DyLight 649 (used to label each protein) for each condition are shown in a spectrum from lowest (violet) to highest (red) signal (gray indicates poor quality data excluded from analysis). Colored bars on left separate sequences into regions of interest, and show consensus identities for all sequences in that region. (b) Insets show examples of binding equilibrium data displaying association and dissociation regimes for P-STAT1 and Myc/Max used to generate the equilibrium binding intensities. Each trace shows binding to a different sequence on the array. (c) The addition of a 10-fold molar excess of U-STAT1 to P-STAT1 does not change the binding preferences of P-STAT1. Representative examples shown from experiments where P-STAT1, 25 nM, with (light colors) or without (dark colors) a 20 min pre-incubation with 250 nM U-STAT1, was tested across an array of STAT recognition sequences. No significant differences in binding preference or affinity were observed.

Figure 2.

The inhibition of STAT1–DNA complexes but not Myc/Max–DNA complexes show sequence-dependent effects. (a) Inhibitor compound LLL3 and analogs were incubated with P-STAT1 and affect equilibrium binding intensity of P-STAT1 to DNA in a sequence-dependent manner (20 μM compound and 50 nM P-STAT1). IC₅₀ for LLL3 was shown to vary in a sequence-dependent manner (Supplementary Figure S2) and ranges from ∼1 μM to >60 μM. ***P-value < 0.001 for optimal GAS site affect to half GAS sites or deviant GAS sites by Student’s t-test.(b) Inhibitor compound Mycro3 was incubated with Myc/Max and affects equilibrium binding intensity in a sequence-independent manner. (c) and (d) Representative data traces showing that the addition of the protein:protein inhibitors cause decreased TF binding.