Clinically relevant dimer interface mutants of STAT1 transcription factor exhibit differential gene expression

Area of Science:

• Molecular Biology
• Genetics
• Cell Signaling

Background:

• Signal transducer and activator of transcription 1 (STAT1) dimerization and conformational changes are crucial for interferon (IFN)-mediated signal transduction.
• The exact molecular mechanisms connecting STAT1 conformational shifts to target gene activation remain incompletely understood.

Purpose of the Study:

• To investigate the functional consequences of two disease-associated STAT1 interface mutants (F172W and T385A) on STAT1 phosphorylation, nuclear accumulation, dephosphorylation, and transcriptional activity.
• To elucidate the role of STAT1 dimerization in regulating specific interferon-stimulated genes.

Main Methods:

• Characterization of STAT1 mutants F172W and T385A in transfected cells.
• In vitro dephosphorylation assays using nuclear phosphatase Tc45.
• Analysis of tyrosine phosphorylation and nuclear accumulation of STAT1.
• Quantitative assessment of endogenous target gene expression (irf1, gpb1, mig1, cxcl10, mcp1).
• DNA-binding assays using fragments with single or "one-and-a-half-GAS" motifs.

Main Results:

• Mutant STAT1 proteins exhibited enhanced tyrosine phosphorylation and prolonged nuclear accumulation upon IFNγ stimulation compared to wild-type STAT1.
• The T385A mutant, similar to F172W, resisted dephosphorylation by Tc45, unlike wild-type STAT1.
• Mutant STAT1 showed significantly enhanced induction of genes (cxcl10, mcp1) with a composite "one-and-a-half-GAS" motif, while genes with a single GAS motif were largely unaffected.
• Mutants displayed increased binding affinity to DNA fragments containing the "one-and-a-half-GAS" motif.

Conclusions:

• Disease-associated STAT1 interface mutants display gene-specific transcriptional effects.
• The study underscores the complex role of STAT1 dimer configuration transitions in regulating gene transcription.
• Mutant STAT1's enhanced binding to specific DNA elements contributes to altered gene expression patterns.