A cell-based bioluminescence assay reveals dose-dependent and contextual repression of AP-1-driven gene expression by BACH2

Sci Rep. 2020 Nov 3;10(1):18902. doi: 10.1038/s41598-020-75732-z.

Abstract

Whereas effector CD4+ and CD8+ T cells promote immune activation and can drive clearance of infections and cancer, CD4+ regulatory T (Treg) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4+ Treg cells and suppressing the effector functions of multiple effector T cell (Teff) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrylamides / pharmacology*
  • Animals
  • Basic-Leucine Zipper Transcription Factors / genetics*
  • Cell Differentiation
  • Dose-Response Relationship, Drug
  • Gene Expression Regulation / drug effects
  • Humans
  • Jurkat Cells
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Measurements / methods*
  • Mice
  • Phenylenediamines / pharmacology*
  • Tetracycline / pharmacology
  • Tetradecanoylphorbol Acetate / analogs & derivatives*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription Factor AP-1 / genetics*

Substances

  • Acrylamides
  • BACH2 protein, human
  • Basic-Leucine Zipper Transcription Factors
  • Phenylenediamines
  • RGFP966
  • Transcription Factor AP-1
  • phorbolol myristate acetate
  • Luciferases
  • Tetracycline
  • Tetradecanoylphorbol Acetate