CHARACTERIZATION OF THE HUMAN MSX-1 PROMOTER AND AN ENHANCER RESPONSIBLE FOR RETINOIC ACID INDUCTION

Previous studies have shown that the expression of some human HOX gene s can be induced by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. However, the mechanisms for the regulation of HOX gene ex pression by RA are still unclear. We have examined the effects of RA o n the human MSX-1 (formerly named HOX-7) gene expression in cultured E C cells (NT2/D1). Furthermore, we have cloned and characterized the hu man MSX-1 promoter and analyzed the activities of the promoter in resp onse to RA. Our results demonstrate that transcription of human MSX-1 is activated by RA in cultured EC cells. This activation is dose and t ime responsive. The MSX-1 promoter was shown to be TATA-box independen t and able to promote transcription in RA-treated EC cells. DNase-I fo otprinting studies revealed protection of several GAGA factor binding sites and an NF-kappa B site upstream to the transcription start site by nuclear extracts prepared from EC cells. A downstream sequence was differentially protected by the nuclear extract from RA treated cells. This differential binding of the sequence with the nuclear extract wa s further confirmed by gel shift assays. This sequence confers to a he terologous promoter with the ability to respond to RA induction. Point mutation within this DNA fragment abolished the binding of the fragme nt to the nuclear extract and the response of this element in a hetero logous promoter to RA induction. Deletion of this enhancer element tog ether with the adjacent NF-kappa B and GAGA sites abolished the abilit y of the promoter to direct transcription in RA-treated EC cells. Howe ver, removal of a downstream DNA fragment from the promoter endowed th e promoter with the ability to direct transcription in RA-untreated ce lls. Taken together, both positive and negative regulatory cis-element s are involved in the regulation of the MSX-1 promoter and coordinate to control the gene expression.