No abstract

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.

Cubitus interruptus (Ci) is a transcriptional factor that is positively regulated by the hedgehog (hh) signaling pathway. Recent work has shown that a 75-kDa proteolytic product of the full-length CI protein translocates to the nucleus and represses the transcription of CI target genes. In cells that receive the hh signal, the proteolysis of CI is inhibited and the full-length protein can activate the hh target genes. Because protein kinase A (PKA) inhibits the expression of the hh target genes in developing embryos and discs and the loss of PKA activity results in elevated levels of full-length CI protein, PKA might be involved directly in the regulation of CI proteolysis. Here we demonstrate that the PKA pathway antagonizes the hh pathway by phosphorylating CI. We show that the PKA-mediated phosphorylation of CI promotes its proteolysis from the full-length active form to the 75-kDa repressor form. The PKA catalytic subunit increases the proteolytic processing of CI and the PKA inhibitor, PKI, blocks the processing. In addition, cells do not process the CI protein to the 75-kDa repressor when all of the PKA sites in CI are mutated. Mutant CI proteins that cannot be phosphorylated by PKA have increased transcriptional activity compared with wild-type CI. In addition, exogenous PKA can increase further the transcriptional activity of the CI mutant, suggesting that PKA has a second positive, indirect effect on CI activity. In summary, we show that the modulation of the hh signaling pathway by PKA occurs directly at the level of CI phosphorylation.Although several signaling cascades have been characterized in detail in multiple organisms, the hedgehog (hh) signaling pathway is of special interest because it plays an important role in the developmental process. The hh gene was first identified by a genetic screen for mutations affecting segment polarity in the Drosophila embryo (1). The hh gene encodes a secreted protein (2), HH, that is involved in pattern formation in both embryogenesis and disc development (3, 4). HH is synthesized in cells located in the posterior compartment of the imaginal discs. The expression of HH is maintained by another secreted protein, Wingless (WG) (2, 5-7), which regulates the expression of a homeodomain transcriptional factor engrailed (en) during early stages of embryo development (8-10). Upon secretion, HH diffuses anteriorly to function in a distance-and concentration-dependent manner to stimulate the transcription of the hh target genes, which include decapentaplegic (dpp), wingless (wg), and patched (ptc) (3,4,11,12). At the membrane level, HH binds to its postulated receptor (ptc), which has at least seven putative transmembrane domains, relieving the inhibition of ptc on smoothened (smo) (13-17). Consequently, smo activates cubitus interruptus (ci), which encodes a transcriptional factor involved in mediating the hh signal from the membrane to the nucleus (18)(19)(20). Additional regulators of the hh pathway include costal2 (cos2), which is believed to tether CI in th...

Each of the homeotic genes of the HOM or HOX complexes is expressed in a limited domain along the anterior‐posterior axis. Each homeotic protein directs the formation of characteristic structures, such as wings or ribs. In flies, when a heat shock‐inducible homeotic gene is used to produce a homeotic protein in all cells of the embryo, only some cells respond by altering their fates. We have identified genes that limit where the homeotic gene Sex combs reduced (Scr) can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is prevented from inducing prothoracic structures by the three bithorax complex (BX‐C) homeotic genes. However, two of the BX‐C homeotic genes, Ultrabithorax (Ubx) and abdominal‐A (abd‐A), have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX‐C gene, Abdominal‐B (AbdB). Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function.

CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signalresponsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatinassociated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.For proper cellular function and the elaboration of developmental programs, gene expression must be regulated tightly. There is increasing evidence that large transcription complexes, composed of unique combinations of sequence-specific activators and repressors, coactivators, and corepressors, play an important role in determining the temporal and spatial patterns of gene expression (for review, see reference 39).The CREB binding protein (CBP) is one of most extensively characterized coactivator proteins. CBP was first identified through its ability to link the cyclic AMP protein kinase (PKA)-phosphorylated form of CREB to components of the basal transcriptional machinery, including TFIIB (14, 34), , and the RNA polymerase II holoenzyme complex (28,44). CBP is highly related to the adenovirus E1A binding protein p300 (17), and CBP and p300 are considered to be functional homologues (4, 38), although a few differences in their activities have been reported (27). CBP and p300 associate with a wide variety of transcriptional activators in addition to CREB, suggesting that each may serve as a transcriptional integrator of different signaling cascades (reviewed in references 20 and 60). Thus, one model for the function of CBP and p300 is bridging DNA binding transcription factors to components of the b...