Mechanisms of gene regulation by the bHLH transcription factor TWIST 2 October 7, 2008

The TWIST2 gene is a transcription factor that belongs to the family of basic helix-loop-helix (bHLH) transcription factors.  Two nonsense mutations (Q119X and Q65X) in TWIST2 code for truncated forms of the transcription factor which are postulated to cause Setleis syndrome (MIM 227260). Setleis syndrome was first described in patients of Puerto Rican ancestry who presented with bilateral temporal marks similar to forceps marks, an aged leonine appearance, absent eyelashes on both lids or multiple rows on the upper lids and none on the lower lids, eyebrows that slanted sharply upward laterally, chin clefting and a rubbery feel of the nose and chin. All affected persons traced their ancestry to the towns of San Sebastian and Aguadilla (Setleis et al, 1963, Marion et al., 1987). No chromosomal abnormalities were found in these and other SS patients (Setleis et al, 1963, and many others). 

TWIST 2 is a Basic-Helix-Loop-Helix (bHLH) Transcription Factor of 160 Amino Acids. The Q119X (PR) mutation results in a loss of the C-terminal domain, and the Q65X (OM) mutation results in the loss of the basic region, HLH, and C-terminal domain . Basic helix-loop helix proteins bind DNA as homo or heterodimers. TWIST2 Is Highly Expressed In developing dermis, cranial mesenchymal cells around the nose, pharyngeal arches and tongue.

Microarray analyses have been used to identify genes potentially regulated by the Setleis syndrome gene (TWIST 2) in human skin fibroblast cultures. Genes found to be differentially regulated in these cells include various genes involved in the inflammatory response, TGFb signaling, bone and skin growth and development. One of these genes is Chordin like-1 (CHRDL1), proposed to modulate the Bone morphogenetic protein (BMP) signaling pathway during formation of the neural plate. The objective of my project is to confirm Twist2 binding to elements in CHRDL1 regulatory regions (-3kb). We are interested in knowing if Twist2 regulates CHRDL1 directly and what is the mechanism it uses. This will be demonstrated performing a reporter gene assay. We expect TWIST 2 to interact directly with CHRDL1 regulatory regions suppressing the expression of this gene.

My role in this research project will be

·         Preparing constructs of the CHRDL1 regulatory regions (-3kb) and cloning them to the pGL4 reporter vector.

·         Transfect Hela cells that will be used in the reporter gene assay. 

·         Optimizing conditions for the Hela transfection.