Modulation of JunD.AP-1 DNA binding activity by AP-1-associated factor 1 (AF-1)

Area of Science:

• Molecular Biology
• T-cell immunology
• Protein-DNA interactions

Background:

• Activating Protein-1 (AP-1) transcription factors regulate gene expression in T-cells.
• JunD is a key component of AP-1 dimers involved in T-cell activation.
• Mechanisms modulating AP-1 DNA binding affinity require further elucidation.

Purpose of the Study:

• To identify and characterize novel factors that modulate AP-1 DNA binding.
• To investigate the role of AP-1-associated factor 1 (AF-1) in regulating JunD-AP-1 complex formation.

Main Methods:

• Partial purification of AF-1 from MLA 144 T-cell nuclear extracts using chromatography.
• DNA-binding assays to analyze AF-1 and JunD interactions.
• DNA cleavage footprinting to assess complex-DNA contacts.

Main Results:

• AF-1, a 34-kDa complex of low molecular mass polypeptides, was partially purified.
• AF-1 dramatically enhances JunD dimer assembly onto AP-1 sites.
• AF-1 increases JunD DNA binding affinity over 100-fold and nearly doubles DNA contacts.
• AF-1 interacts differentially with Jun homodimers and Jun.Fos heterodimers.

Conclusions:

• AF-1 is a novel, protein-specific modulator of AP-1.JunD activity in T-cells.
• AF-1 facilitates rapid formation of a tri-molecular complex with JunD and DNA.
• AF-1 represents a significant regulatory mechanism for AP-1 transcription factor function.