title: Proteomic analysis yields an unexpected trans-acting point in control of the human sympathochromaffin phenotype.
abstract: BACKGROUND
  The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release-inhibitory fragment. Because genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene.
  
  
  RESULTS
  Bioinformatic analysis suggested that the common G-462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPARγ-RXRα. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G-462A. On oligonucleotide affinity chromatography followed by electrospray ionization followed by 2-dimensional (tandem) mass spectrometry analysis of A allele eluates, the transcription factor LEF1 (lymphoid enhancer-binding factor-1) was identified. Interaction of LEF1 with the A allele at G-462A was confirmed by supershift. On cotransfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G-462A element fused to a heterologous (SV40) promoter. Because β-catenin (CTNNB1) can heterodimerize with LEF1, we tested the effect of cotransfection of this factor and again found A allele-specific perturbation of CHGA transcription.
  
  
  CONCLUSIONS
  Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by supershift. Coexpression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.
authors.affiliation: Department of Medicine and Institute for Genomic Medicine, University of California at San Diego, La Jolla, 92093–0838, USA.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Chiron
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Wei
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Chen
authors.forename: Kuixing
authors.initials: K
authors.lastname: Zhang
authors.forename: Gen
authors.initials: G
authors.lastname: Wen
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Fischer
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Mahata
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D002838:Chromaffin System; D053379:Chromogranin A / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D040901:Proteomics; D013564:Sympathetic Nervous System; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: C506150:CHGA protein, human; D053379:Chromogranin A; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1