Role of the TWIST proteins and ADD1 in the regulation of the CHRDL1 gene

Abstract

Puerto Rican Setleis Syndrome (SS) patients harbor a nonsense mutation (TWIST2‐Q119X) that causes truncation of the C‐terminal region required for transactivation of target genes, resulting in facial and skin abnormalities. We found that the chordin‐like 1 (CHRDL1) gene is up‐regulated in SS patients and silencing of TWIST2 causes an increase in CHRDL1 gene expression. CHRDL1 codes for a bone morphogenic protein antagonist expressed in mesenchymal and neural tissues. Putative TWIST binding sites were found at positions −2421, −2051, −1000, −763, and +46 relative to the TSS of the CHRDL1 gene. EMSA analysis showed specific binding activity of TWIST1 and TWIST2 homodimers to the −2421 putative site, which TWIST2‐Q119X could not bind. An adjoining site was bound by SREBP1c/ADD1, a transcription activator inactivated by TWIST2. EMSA analysis suggests that TWIST2 and ADD1 compete for binding the same − 2421 site. Luciferase reporter assays revealed that the 3000bp CHRDL1 gene upstream region drives its expression and ADD1 increases it 2.6X over basal levels. TWIST2, but not Q119X, was able to block activation by ADD1, while overexpression of TWIST2 Q119X results in increased expression. We suggest that the TWIST2 Q119X mutant protein cannot bind to the −2421 site nor compete with ADD1, but may sequester other transcription factors important for CHRDL1 repression. Supported by NIH Grants G12RR03051 and R25GM061838