Abstract
Purpose: :
Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is known to be involved in eyelid closure during mouse embryonic development. A temporal-spatial MAP3K1expression pattern is found in the leading edge of the eyelid epithelium just prior to the onset of eyelid closure. We hypothesize that the induction of MAP3K1 expression is a critical for the control of embryonic eyelid closure. The purpose of this study is to investigate the molecular mechanisms involved in Map3k1 promoter activation.
Methods: :
Bioinformatic approaches were used to predict the promoter region of mouse Map3k1. A DNA fragment, 1900 bp from the transcription start site (TSS) of Map3k1 encompassing the predicted promoter sequences, was isolated using polymerase chain reaction (PCR). Deletion and truncation mutants of this fragment were cloned into pGL3, a promoter-less expression vector containing a luciferase (Luc) reporter gene. The promoter activities were monitored in HEK293 cells following transient transfection. Chromatin immunoprecipitation (ChIP) was used to analyze the association of MAP3K1 with the Map3k1 Proximal promoter. For measuring endogenous MAP3K1 expression, we isolated mouse embryonic fibroblasts (MEFs) from the Map3k1ΔKD fetuses, in which a β-galactosidase gene was knocked in the Map3k1 locus and thus β-gal expression was under the control of the Map3k1 promoter. These cells were used to study the induction of endogenous Map3k1 promoter activity following adenoviral infections with both wild type (WT) and kinase inactive (KM) forms of MAP3K1.
Results: :
Infection of Map3k1ΔKD MEFs with adenoviral MAP3K1 and cotransfection of MAP3K1 with the p1900Map3k1-Luc fragment in 293 cells showed a 2-fold increase of the endogenous (β-galactosidase) promoter and a 4-fold induction of the exogenous (Luc) Map3k1 promoter activities. MAP3K1 was also found to be associated with the proximal promoter in the ChIP assay.
Conclusions: :
Our in vitro studies show that MAP3K1 can positively regulate its own promoter activities. Since kinase-active and -inactive forms of MAP3K1 have a similar effect, we suggest that the auto-regulatory role of MAP3K1 is independent of its kinase activity.
Keywords: eyelid • gene/expression • signal transduction