Interactive Fly, Drosophila

Regulation of FGF transcription

The mechanisms controlling the proliferation of astrocytes are of great interest but are not well defined. The endogenous neuropeptides, endothelin-3 (ET-3), and atrial natriuretic peptide (ANP), modulate the proliferation of astrocytes by positively and negatively regulating the transcription of the immediate-early gene egr-1 (Drosophila homolog: Stripe) which transactivates basic fibroblast growth factor (bFGF) by unknown mechanisms. In these studies, the involvement of MAP kinase (Erk) activation by ET-3 in the transcription of egr-1 (Drosophila homolog: Stripe), and the molecular determinants by which Egr-1 transactivates bFGF were determined. Transfection of astrocytes with a mitogen-activated protein (MAP) kinase (MAPK) expression vector increased the transcription of a cotransfected egr-chloramphenicol acetyltransferase (CAT) construct 3-fold. This induction is totally abolished by a dominant negative MAPK mutant. A 3-fold induction of egr-CAT expression by ET-3 is significantly reduced by treatment with ANP, or a cotransfected dominant negative MAPK plasmid. Using mobility shift assays, it has been shown that ET-3 induces the expression of Egr-1 protein that binds specifically to several early growth-related protein (Egr-1) binding sites on the bFGF promoter, and that this effect is significantly reversed by treatment with ANP. The Sp1 transcriptional factor is bound at these same sites, but is not stimulated by ET-3. Deletion experiments indicate that only the site at -160 bp of the bFGF promoter is significant for bFGF transactivation by Egr-1. It is concluded that the astrocyte mitogen, ET-3, stimulates egr-1 transcription through a MAP kinase (Erk) related mechanism, and that Egr-1 transactivates bFGF through a specific, noncanonical, Egr-1 site on the promoter. ANP inhibits each of these steps, providing a pathway for its anti-proliferative action (Biesiada, 1997).

Basic fibroblast growth factor (bFGF) is an important growth factor for neuroectoderm- and mesoderm-derived cells. In addition bFGF is an important angiogenic factor and appears to contribute to tumorigenesis. This is exemplified by the fact that bFGF is expressed in a large majority of human gliomas and that bFGF expression is critical for the growth and tumorigenesis of these cells. It has been shown previously that bFGF can induce its own expression through an increase in bFGF mRNA. bFGF leads to its own synthesis through an autoregulated transcriptional response that requires the transcription factor Egr-1 (also known as Krox24, Zif268 and NGFI-A). Egr-1 binds to two DNA elements in the bFGF promoter and positively regulates transcription. Mutation of these sites blocks the ability of bFGF to transcriptionally regulate the bFGF promoter. These data indicate a mechanism to explain how bFGF functions to autoregulate its expression and demonstrate that Egr-1 is as an essential transcription factor in this process (Wang, 1997).

In pre B cell leukemias containing the chromosome translocation t(1;19), Pbx1 (a homolog of Drosophila Extramachrochaetae) is converted into a strong transactivator by fusion to the activation domain of the bHLH transcription factor E2A (a homolog of Drosophila Daugherless). The E2A-Pbx1 fusion protein should therefore activate transcription of genes normally regulated by Pbx1. The subtractive process of representational difference analysis was used to identify targets of E2A-Pbx1. E2A-Pbx1 can directly activate transcription of a novel member of the fibroblast growth factor family of intercellular signaling molecules, FGF-15. FGF-15 is the most divergent in sequence from other known FGF family members. All FGF's share a conserved central region of approximately 120 amino acids which in FGF-1 and FGF-2 consists of 12 antiparallel beta strands that form a structure with threefold internal symmetry, called a beta-trefoil. Nine of these beta strands contain a highly conserved hydrophobic core residue. Eight of these hydrophobic residues are present in FGF-15. FGF-15 is between 15 and 30% identical to the other family members, with the greatest similarity to FGF-7 and FGF-1 and the least similarity to FGF-8. The FGF-15 gene is expressed in a regionally restricted pattern in the developing nervous system, suggesting that FGF-15 may play an important role in regulating cell division and patterning within specific regions of the embryonic brain, spinal cord and sensory organs (McWhirter, 1997).

Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture, FGF-4 expression is restricted to undifferentiated embryonic stem cells and embryonal carcinoma (EC) cell lines. EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that binds adjacent sites on the FGF-4 enhancer. This latter activity is carried out by Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis (Yuan, 1995).

Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. Fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. Increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which fail to activate transcription are also compromised in their ability to form the Oct* complexes even though they can still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes (Ambrosetti, 1997).

Embryonic development requires a complex program of events that are directed by a number of signaling molecules whose expression must be rigorously regulated. Expression of Fgf4, which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in embryonal carcinoma (EC) cells by the synergistic interaction of Sox2 and Oct-3 with the Fgf4 EC cell-specific enhancer. To verify whether this mechanism is also operating in vivo, and to identify new elements controlling Fgf4 gene expression in distinct developmental stages, the expression of LacZ reporter plasmids containing different fragments of the Fgf4 gene have been analyzed in transgenic mouse embryos. Utilizing these transgenic constructs Fgf4 gene expression could be recapitulated, for the most part, during embryonic development. Most of the cis-acting regulatory elements determining Fgf4 embryonic expression are located in conserved regions within the 3' UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct-proteins binding sites. Specific and distinct enhancer elements could be identified that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Evidence is also presented that the very restricted pattern of expression of Fgf4 transcripts in the AER results from the combined action of positive and negative regulatory elements located 3' to the Fgf4 coding sequences. Thus the Fgf4 gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression (Fraidenraich, 1998).

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. Cultures of human astrocytes were used to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increases the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs have identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identifies a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicate that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identifies 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: (1) an initial induction by reduced cell contact, and (2) further activation, by either growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively (Moffett, 1998).

Fibroblast growth factor-8 (Fgf8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. This study reports the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 contains promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 enhances 3-4 fold the human growth hormone expressed from the Fgf8 promoter, in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments document the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrate that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicates that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment (Gemel, 1999).

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, is not affected in mice deficient for the IIIb isoform. FgfR2-IIIb ^-/-lacZ mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence does not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but does prevent induction of Shh and Fgf4, indicating that these genes are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. It is proposed that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth (Revest, 2001).

The respiratory primordium is positioned and its territory is defined in the foregut. The visceral mesoderm of the respiratory primordium acquires the inducing potential that is necessary for endodermal budding morphogenesis and respiratory endoderm formation. Tbx4, a member of the T-box transcription factor gene family, is specifically expressed in the visceral mesoderm of the lung primordium. To analyze the function of Tbx4, it was ectopically expressed in the visceral mesoderm of the foregut using in ovo electroporation. Ectopic Tbx4 induces ectopic bud formation in the esophagus by activating the expression of Fgf10. Conversely, interference of Tbx4 function results in repression of Fgf10 expression and in failure of lung bud formation. In addition, ectopic Tbx4 or Fgf10 also induces ectopic expression of Nkx2.1, a marker gene specific for the respiratory endoderm, in the underlying esophagus endoderm. When the border of the Tbx4 expression domain, which demarcates the respiratory tract and the esophagus, is disturbed by misexpression of Tbx4, formation of the tracheo-esophageal septum fails. These results suggested that Tbx4 governs multiple processes during respiratory tract development; i.e. the initial endodermal bud formation, respiratory endoderm formation, and septation of the respiratory tract and the esophagus (Sakiyama, 2002).

The zinc finger transcription factor GLI3 is considered a repressor of vertebrate Hedgehog (Hh) signaling. In humans, the absence of GLI3 function causes Greig cephalopolysyndactyly syndrome, affecting the development of the brain, eye, face, and limb. Because the etiology of these malformations is not well understood, the phenotype of mouse Gli^-/- mutants was examined as a model to investigate this. An up-regulation of Fgf8 is observed in the anterior neural ridge, isthmus, eye, facial primordia, and limb buds of mutant embryos, sites coinciding with the human disease. Intriguingly, endogenous apoptosis is reduced in Fgf8-positive areas in Gli^-/- mutants. Since SHH is thought to be involved in Fgf8 regulation, Fgf8 expression was compared in Shh^-/- and Gli^-/-;Shh^-/- mutant embryos. Whereas Fgf8 expression is almost absent in Shh^-/- mutants, it is up-regulated in Gli^-/-;Shh^-/- double mutants, suggesting that SHH is not required for Fgf8 induction, and that GLI3 normally represses Fgf8 independently of SHH. In the limb bud, evidence is provided that ectopic expression of Gremlin in Gli^-/- mutants might contribute to a decrease in apoptosis. Together, these data reveal that GLI3 limits Fgf8-expression domains in multiple tissues, through a mechanism that may include the induction or maintenance of apoptosis. It is concluded that Fgf8 may not be a direct target of GLI3 but that the size of the Fgf domain may be regulated by GLI3 indirectly; when GLI3 is present, it activates the expression of Bmps, which regulates cell death to alter the size of FGF8 domains (Aoto, 2002).

Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, focus was placed on two transcription factors, Oct4 and Sox2, since their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, it was found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. The DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners (Reményi, 2003).

The interaction of Oct1 and Oct4 with Sox2 was investigated on two different DNA enhancers to test whether a previously discovered regulation mechanism of DNA-mediated swapping of the arrangement of homodimers may also be applicable for unrelated transcription factor assemblies. The crystal structure of the ternary Oct1/Sox2/FGF4 enhancer element complex was solved and then homology modeling tools were used to construct an Oct4/Sox2/FGF4 as well as an Oct4/Sox2/UTF1 structural model. These models reveal that the FGF4 and the Undifferentiated Transcription Factor 1 (UTF1) enhancers mediate the assembly of distinct POU/HMG complexes, leading to different quaternary arrangements by swapping protein-protein interaction surfaces of Sox2. Moreover, it has been demonstrated that Sox2 uses one of its two protein interacting surfaces to assemble a ternary complex with another unrelated transcription factor on a late-embryonic-stage-specific enhancer (Pax6/Sox2 on the DC5 element). These findings outline a simple mechanism for promiscuous yet highly specific assembly of transcription factors, in which the sequence of DNA enhancers governs a combinatorial use of redundant protein-protein interaction surfaces (Reményi, 2003).

Gene regulation downstream of FGF

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. The nature of Fgf response element (FRE) has been investigated in a posterior neural gene, Xcad3 (Xenopus caudal homolog), which plays a crucial role of posterior neural development. Evidence suggests that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s) (Haremaki, 2003).

The reporter constructs containing the FREs exhibit high dose dependence on Fgf similar to that shown for endogenous Xcad3, when examined in the embryonic cell culture assay. Sequence and mutagenesis analyses reveal that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs respectively) that lie in juxtaposition. The EBM is known to serve as the binding site for Ets family transcription factors that are nuclear effectors of the Fgf/Ras/Mapk pathway. Indeed, functional and physical analyses show that Ets proteins are involved in the Fgf response of Xcad3 as transcriptional activators, and that Xcad3 is directly targeted by the Fgf signaling pathway. This conclusion is consistent with the previous observation that Fgf can induce Xcad3 expression in the animal cap assay within 2 hours of its addition and even in the presence of the protein synthesis inhibitor cycloheximide, which indicates that Xcad3 is an immediate early target of Fgf signaling (Haremaki, 2003 and references therein).

TLBMs could serve as the binding sites for Tcf/Lef family transcription factors that are nuclear effectors of the Wnt/ß-catenin pathway. It was anticipated that XTcf3 would functioned as a co-activator of Ets proteins, since Wnt signaling has been suggested as being involved in activation of posterior neural genes. Surprisingly, however, functional analysis reveals that XTcf3 acts as a repressor of Xcad3. The data suggest that the endogenous pool of ß-catenin in ectoderm cells is considerably smaller compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in turn implies that Wnt signaling could activate Xcad3 expression in embryonic cells, when they are provided with a larger pool of ß-catenin. Marginal zone cells of the early gastrula embryo, where Xcad3 is initially expressed, are among such candidate cells, since a relatively large amount of ß-catenin is translocated into the nucleus in these cells. Recently, a mutant function of Tcf3 as a repressor has revealed in the zebrafish headless mutant that carries a mutation in Tcf3. In this mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2 is de-repressed in more anterior neural region, leading to severe head defects. It would be interesting to know whether similar anterior expansion is seen in Cdx gene expression in this mutant (Haremaki, 2003 and references therein).

Sox2 is de-repressed by Bmp antagonists in the neurogenic region of ectoderm during neural induction. Sox2, which shares a cognate DNA bindings motif with Tcf/Lef family members, is required as a co-activator for the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for TLBMs in the composite FREs to cooperate with Ets proteins that bind to adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family transcription factors interact with specific partner factors to direct signals to target genes, but direct partnership between them has not been reported. Collectively, these results indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on the FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox proteins and negatively acting Tcf protein (Haremaki, 2003).

branchless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

The Interactive Fly resides on the
Society for Developmental Biology's Web server.