Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3.

Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to hedgehog protein (Hh) signaling. In the anterior region, in contrast, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products (GLIs) 1, 2, and 3) were identified, but their functional difference in Hh signal transduction is unknown. Here, we report distinct roles for GLI1 and GLI3 in Sonic hedgehog (Shh) signaling. GLI3 containing both repression and activation domains acts both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. The trans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to the Gli1 promoter and induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the activation of the target gene Gli1.

Drosophila CBP is required for dorsal-dependent twist gene expression.

Although CREB-binding protein (CBP) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Recently, we isolated a Drosophila CBP (dCBP) mutant, and found dCBP to be maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal dorsal (dl) protein (Dl; refs 8-10), a transcription factor that is homologous to the NF-kappa B family of proteins. The activity of dl is negatively regulated by cactus (cact), a Drosophila homologue of I kappa B. Here, we show that dCBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for dl-mediated activation of the twi promoter.

Trans-activation by the Drosophila myb gene product requires a Drosophila homologue of CBP.

Attempts to demonstrate trans-activation activity by the Drosophila myb gene product (D-Myb) have been unsuccessful so far. We demonstrate that co-transfection of Schneider cells with a plasmid expressing the Drosophila homologue of transcriptional co-activator CBP (dCBP) results in transactivation by D-Myb. Using this assay system, the functional domains of D-Myb were analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution.

Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling.

The transcription factor CBP, originally identified as a coactivator for CREB, enhances transcription mediated by many other transcription factors. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haploinsufficiency disorder characterized by abnormal pattern formation, but the mechanism by which decreased CBP levels affect pattern formation is unclear. The hedgehog (hh) signalling pathway is an important determinant of pattern formation. cubitus interruptus (ci), a component in hh signalling, encodes a transcription factor homologous to the Gli family of proteins and is required for induction of the hh-dependent expression of patched (ptc), decapentaplegic (dpp) and wingless (wg). Haploinsufficiency for the ci-related transcription factor Gli3 causes phenotypic changes in mice (known as 'extra-toes) and humans (Greig's cephalopolysyndactyly syndrome) that have similarities to Rubinstein-Taybi syndrome. Here we show that Drosophila CBP (dCBP) functions as a coactivator of Ci, suggesting that the dCBP-Ci interaction may shed light on the contribution of CBP to pattern formation in mammals.

CBP as a transcriptional coactivator of c-Myb.

CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A). CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form. We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator. CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro. The domain of CBP that touches c-Myb is also required for binding to CREB. A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay. CBP stimulates the c-Myb-dependent transcriptional activation. Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation. In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation. Our data thus identify CBP as a coactivator of c-Myb. These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously.

Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages.

A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.

Identification of a different-type homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations in Drosophila.

The Bar mutation B of Drosophila melanogaster and optic morphology mutation Om(1D) of Drosophila ananassae result in suppression of ommatidium differentiation at the anterior portion of the eye. Examinations was made to determine the genes responsible for these mutations. Both loci were found to share in common a different type of homeobox gene, which we call "BarH1." Polyptides encoded by D. melanogaster and D. ananassae BarH1 genes consist of 543 and 604 amino acids, respectively, with homeodomains identical in sequence except for one amino acid substitution. A unique feature of these homeodomains is that the phenylalanine residue in helix 3, conserved in all metazoan homeodomains so far examined, is replaced by a tyrosine residue. By Northern blotting, considerably more BarH1 RNA was detected in the Bar mutant than in wild type. P element-mediated transformation showed Bar-like eye malformation to be induced by transient overexpression of the BarH1 gene in the late third-instar larvae. Somatic recombination analysis indicated normal gene functions of the Bar region, including the BarH1 gene, to be required for normal eye morphogenesis.

DNA binding activity of the BarH1 homeodomain of Drosophila.

Using a series of deletion mutants of BarH1, a Drosophila homeobox gene required for eye morphogenesis, the DNA-binding region of the BarH1 protein was determined. Not only homeodomain but also its upstream sequence were found to be necessary for binding, whereas about a half of the conserved downstream sequence (Bar domain) was dispensable.

Positive and negative regulation of transcription by a cleavage product of Ada protein.

The 39,000 Mr Ada protein of Escherichia coli that carries two distinct methyltransferase activities and activity to promote transcription of the ada and the alkA genes is cleaved by a cellular proteinase. As a result, the 20,000 and the 19,000 Mr proteins are formed, which are derived from the N-terminal and the C-terminal halves of the protein, respectively. To elucidate the molecular mechanism of transcriptional control by Ada protein, the N-terminal 20,000 Mr protein was overproduced by manipulating the cloned ada gene. The protein possessed an activity to transfer a methyl group from the methylphosphotriester of the alkylated DNA to its own molecule and retained the potential to promote transcription of the alkA gene. The methylated form of the 20,000 Mr proteins binds to the proper alkA regulatory sequence, as does the intact Ada protein, and facilitates further binding of RNA polymerase to the promoter, thus forming an active transcription initiation complex. The non-methylated 20,000 Mr protein was incapable of binding itself or supporting RNA polymerase binding to the alkA promoter. When the 20,000 Mr protein was produced under the control of the lac promoter in E. coli and then exposed to a methylating agent, a considerable amount of 3-methyladenine-DNA glycosylase II, the product of the alkA gene, was formed. Thus, the results obtained in in vitro experiments were confirmed by the events observed in vivo. The methylated 20,000 Mr protein also binds to the ada promoter; however, it does not facilitate further binding of RNA polymerase to the promoter nor does it promote ada transcription in vitro. These findings indicate that the N-terminal half of Ada protein is mainly responsible for recognition of and binding to alkA and the ada regulatory sequence. The methylated 20,000 Mr protein occupies the same region of the ada promoter to which the intact Ada protein would bind, thereby suggesting that it acts as a repressor for expression of the ada gene. The ada transcription promoted by the Ada protein was greatly inhibited by the methylated, but not the non-methylated, form of the 20,000 Mr protein. In an in vivo system, formation of the 20,000 Mr protein leads to inhibition of transcription from the ada promoter. We suggest that termination of the adaptive response may come about by proteolytic cleavage of the Ada protein, the result being a loss of the activator as well as formation of the repressor for ada transcription.