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The bHLH protein Twist2 regulates expression of the CHRDL1 (Chordin‐like 1) gene in human skin fibroblasts
Author(s) -
Casasnovas Jose J,
Franco Hector L,
Cadilla Carmen L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.660.18
Subject(s) - microbiology and biotechnology , gene knockdown , transactivation , transfection , gene , microarray analysis techniques , hek 293 cells , biology , electrophoretic mobility shift assay , chemistry , transcription factor , gene expression , genetics
Puerto Rican Setleis Syndrome (SS) patients harbor a nonsense mutation in Gln 119 of TWIST2 and have craniofacial abnormalities. This mutation causes truncation of the C‐terminal, which is required for transactivation. Microarray analysis and real time RT‐PCR of normal and patient skin fibroblasts revealed 287 differentially regulated genes, where the chordin‐like 1 (CHRDL1) gene being up‐regulated in SS patients. CHRDL1 is a bone morphogenic protein (BMP) antagonist which is expressed in mesenchymal and neural tissues. Knockdown of Twist2 by siRNA in SV40‐transformed human fibroblasts showed increased levels of CHRDL1 transcripts 48 hrs after transfection. Additionally, transfection of wild type Twist2 in SS patient fibroblasts reduced CHRDL1 expression. In silico analysis of 3000 bp upstream from the transcription start site of the CHRDL1 gene identified putative TWIST binding sites on positions ‐2663, ‐2344, ‐1292, ‐1055, and ‐247. DNA sequence analysis carried out using mVISTA showed conservation of these sites. Electrophoresis mobility shift assay (EMSA) showed specific binding activity of Twist1 and Twist2 homodimers to the ‐2663 putative site while the Q119X protein weakly bound the ‐2663 putative site. This difference in binding could account for CHRDL1 being upregulated in patients. The MBRS RISE R25GM061838 and RCMI G12RR03051 grants provided support for this work.