Regulatory mechanisms governing FGF-4 gene expression during mouse development
✍ Scribed by Claudio Basilico; Davide Ambrosetti; Diego Fraidenraich; Lisa Dailey
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 167 KB
- Volume
- 173
- Category
- Article
- ISSN
- 0021-9541
No coin nor oath required. For personal study only.
✦ Synopsis
protein-binding motif (7) (Fig. 1). Using a DNA probe Embryonic development entails a complex program spanning the octamer sequence and F9 nuclear exof events that must be executed in a precise and ordered tracts, we determined that the octamer element bound fashion. Much of the coordination of these events is either Oct 1 or Oct 3, whereas an additional F9-specific achieved by the directive instructions of extracellular activity, originally designated as Fx, interacted with signaling molecules that serve to activate or repress sequences immediately upstream of the Oct site (Fig. transcription of specific gene subsets and ultimately 2). The DNA site bound by Fx corresponds to a functioninfluence the proliferative state or identity of the target ally important sequence identified by the mutation cell. Because these signaling molecules have a profound analysis (m12, Fig. 1). Furthermore, Fx also was found effect on cell growth and differentiation, it is imperative to form ternary complexes (Oct-1* or Oct-3*) on the that the expression of these molecules be rigorously FGF-4 enhancer DNA probe with octamer binding proand specifically regulated. Thus, elucidation of the regteins ( 7). The functional importance of the Oct* complex ulatory mechanisms that govern differential expression was suggested by the observation that the ability of of these signaling molecules is fundamental to an undifferent enhancer mutants to form the Oct* complexes derstanding of the process of development.
correlates with enhancer function (data not shown). Be-Our work has focused on deciphering the regulatory cause these observations pointed to a fundamental role mechanisms that underly differential gene transcripfor Fx and octamer-binding proteins in activation of the tion of one such signaling molecule, fibroblast growth FGF-4 enhancer, we sought to identify and characterfactor 4 (FGF-4). FGF-4 was originally identified as ize Fx. an oncogene capable of transforming fibroblast cells in
The binding of Fx to the DNA probe is completely tissue culture (1,2). Subsequently, FGF-4 has been eliminated in the presence of poly dI-dC (Fig. 2). This shown to play essential roles in different developmental observation indicated that Fx must bind primarily with stages including viability of the postimplantation blas-dAdT residues within the DNA minor groove. This tocyst and, at later stages, in outgrowth and patterning property and the apparent recognition sequence of Fx, of the developing limb (3,4). Thus, discrete, temporally TTCTTTGT, suggested to us that Fx might be a memand spatially restricted expression of the FGF-4 gene ber of the Sox factor family of developmental regulators is essential to proper development.
(8,9). Based on this prediction, we exploited the fact To identify the cis and trans-acting elements that that the Sox factors each contain a highly conserved direct expression of the FGF-4 gene, we took advantage DNA binding domain, called the HMG domain. Thereof our previous observation that, in addition to its exfore, we generated the Sox family HMG box by polymerpression in the embryo, the FGF-4 gene is transcribed ase chain reaction using cDNA derived from F9 cells in embryonic stem cells and embryonal carcinoma (EC) and degenerate oligonucleotide primers complemencell lines, such as F9, in tissue culture (5). In contrast, tary to the most conserved portions of this HMG do-FGF-4 expression is downregulated after treatment of main. Using this approach, we isolated several HMG EC cells with retinoic acid and is not observed in Hela box DNA fragments, all of which corresponded to that or NIH3T3 cells (5). Using a variety of reporter plaspreviously described for Sox2 (10). The Sox2 HMG box mids in which the CAT gene was under the control of DNA fragment was used as a probe to isolate a fullthe FGF-4 promoter in transfection assays, we identilength Sox2 cDNA clone from an F9 cDNA library. The fied an enhancer element present in both the murine murine clone Sox2 cDNA contains an open reading and human FGF-4 genes that can confer F9 specific frame for a protein of 318 amino acids with, a predicted transcriptional activity (6). Both the location in the nontranslated portion of the third exon and the sequence of the enhancer are conserved between the mu-Contract grant sponsor: National Cancer Institute; Contract rine and the human gene. The analysis of enhancer grant number: PHS CA42568; Contract grant sponsor: PEW Latin American Fellows Program. base substitution mutants after transfection of F9 cells revealed that multiple elements are required for full *Correspondence to: Claudio Basilico, Department of Microbiol- enhancer activity, including a putative Sp1 binding site ogy and Kaplan Cancer Center,