Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter

Nucleic Acids Res. 2018 Jan 25;46(2):929-941. doi: 10.1093/nar/gkx1183.

Abstract

Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (-794 CATT5-8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • Fluorescence Polarization
  • Gene Expression Regulation
  • Genetic Predisposition to Disease / genetics
  • Genetic Predisposition to Disease / prevention & control
  • Humans
  • Intramolecular Oxidoreductases / genetics*
  • Intramolecular Oxidoreductases / metabolism
  • Macrophage Migration-Inhibitory Factors / genetics*
  • Macrophage Migration-Inhibitory Factors / metabolism
  • Microsatellite Repeats / genetics*
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism
  • Polymorphism, Genetic
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • Transcription Factor Pit-1 / chemistry
  • Transcription Factor Pit-1 / genetics*
  • Transcription Factor Pit-1 / metabolism

Substances

  • Macrophage Migration-Inhibitory Factors
  • Oligonucleotides
  • Transcription Factor Pit-1
  • DNA
  • Intramolecular Oxidoreductases
  • MIF protein, human