Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element

J Biol Chem. 2015 Jun 19;290(25):15878-15891. doi: 10.1074/jbc.M114.635144. Epub 2015 Apr 30.

Abstract

Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.

Keywords: ATF3; ATF4; CEBPβ; CHAC1; DNA transcription; DNA-protein interaction; apoptosis; endoplasmic reticulum stress (ER stress); oxidative stress; unfolded protein response (UPR).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Activating Transcription Factor 3 / genetics
  • Activating Transcription Factor 3 / metabolism*
  • Activating Transcription Factor 4 / genetics
  • Activating Transcription Factor 4 / metabolism*
  • Animals
  • Base Sequence
  • CCAAT-Enhancer-Binding Proteins / genetics
  • CCAAT-Enhancer-Binding Proteins / metabolism
  • Endoplasmic Reticulum Stress / physiology
  • Gene Expression Regulation, Enzymologic / physiology*
  • Glutathione / genetics
  • Glutathione / metabolism*
  • HEK293 Cells
  • Humans
  • Mice
  • Oxidative Stress / physiology
  • RNA, Messenger / biosynthesis*
  • RNA, Messenger / genetics
  • Response Elements / physiology*
  • Sequence Deletion
  • gamma-Glutamylcyclotransferase / biosynthesis*
  • gamma-Glutamylcyclotransferase / genetics

Substances

  • ATF3 protein, human
  • ATF4 protein, human
  • Activating Transcription Factor 3
  • CCAAT-Enhancer-Binding Proteins
  • GGCT protein, human
  • RNA, Messenger
  • Activating Transcription Factor 4
  • gamma-Glutamylcyclotransferase
  • Glutathione