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. Author manuscript; available in PMC: 2010 Apr 3.
Published in final edited form as: Biochem Biophys Res Commun. 2009 Feb 11;381(2):241–246. doi: 10.1016/j.bbrc.2009.02.016

FIG. 1. Activation of Hspa1b promoter by the Msx1 and Msx2.

FIG. 1

(A) Schematic of the 650bp Hspa1b promoter-lacZ reporter construct used in transient transfection assays. (B) The Hspa1b promoter responded robustly to the induction by Msx1 and Msx2 in C2C12 cells. (C) Cotransfection of Msx1 with the Hspa1b-reporter construct also resulted in the transcriptional induction of the Hspa1b promoter in αTN4 mouse epithelial cell line. (D) The Hspa1b promoter showed dose-dependent response to Msx1. (E) A series of promoter deletion constructs (left) was made to map cis regulatory sequences that responded to Msx induction (right). A 205bp Hspa1b promoter fragment was found to be sufficient in responding to the induction by the Msx1. C2C12 cells were co-transfected with 100 ng of individual Hspa1b deletion-reporter plasmids and 300 ng Msx1-3xFlag expression plasmids or an empty control plasmid. Beta-galactosidase activities were normalized to an internal control expressing luciferase. Each transfection was performed in triplicates with the standard error shown. (F). A schematic representations of Hspa1b promoter reporter deletion constructions. HSE1 and HSE2 corresponding to Heat shock factor binding sites and a Msx binding site are shown. Mutations introduced into the HSE2 site are indicated by lower-case letters. The striped rectangle denotes the position of the TATA box. (G) The Msx binding site is not required for the induction of Hspa1b promoter activity by Msx1. Removal of the Msx consensus binding site didn’t affect the transcriptional response of the Hspa1b promoter as transcriptional activities between the Hsp-160 and Hsp-150 failed to show significant difference. (H) Cotransfection of a non-DNA binding Msx1 mutant (Msx1-A) did not alter the ability of the mutant protein to transactivate the 205bp Hspa1b promoter-reporter. All transfections were performed in triplicates. (I). Deletion of the distal HSE (Hsp-180) lead to a 50% reduction in Msx1-stimulated transcriptional activity; removal of both HSEs (Hsp150-120) or mutagenized HSE2 (Hsp-150hm) abolished the Msx1-dependent promoter activity. All transfections were performed in triplicates.