Heterochromatin-mediated gene silencing is not affected by Drosophila CBP activity.

Cyclic AMP Response Element Binding protein (CREB)-binding protein (CBP) is an acetyltransferase important for modifying histones and chromatin-associated proteins and thus affecting transcription and other DNA metabolic processes. We found that the Drosophila CBP (dCBP) is associated with the NAD(+)-dependent deacetylase, SIR2, which was originally identified as a silencing information regulator in yeast that models silenced and repeated sequence chromatin such as centric heterochromatin, telomeres, and the repeated rDNA sequences. As in yeast, Drosophila sir2 (dsir2) affects the formation and/or function of centric heterochromatin. The fact that we found dCBP in immunecomplexes with dSIR2 in vivo and found that dCBP can interact with dSIR2 directly in vitro suggested that dCBP might affect the packaging of silencing heterochromatin as well. A careful study of the dCBP mutations provides evidence that dCBP does not affect the formation and/or function of centric heterochromatin and thus may affect other dSIR2 functions.

Ras-independent activation of ERK signaling via the torso receptor tyrosine kinase is mediated by Rap1.

In Drosophila embryos, the Torso receptor tyrosine kinase (RTK) activates the small G protein Ras (D-Ras1) and the protein kinase Raf (D-Raf) to activate ERK to direct differentiation of terminal structures . However, genetic studies have demonstrated that Torso, and by extension other RTKs, can activate Raf and ERK independently of Ras . In mammalian cells, the small G protein Rap1 has been proposed to couple RTKs to ERKs. However, the ability of Rap1 to activate ERKs remains controversial, in part because direct genetic evidence supporting this hypothesis is lacking. Here, we present biochemical and genetic evidence that D-Rap1, the Drosophila homolog of Rap1, can activate D-Raf and ERK. We show that D-Rap1 binds D-Raf and activates ERKs in a GTP- and D-Raf-dependent manner. Targeted disruption of D-Rap1 expression decreased both Torso-dependent ERK activation and the ERK-dependent expression of the zygotic genes tailless and huckebein to levels similar to those achieved in D-Ras1 null embryos. Furthermore, combined deficiencies of D-Ras1 and D-Rap1 completely abolished expression of these genes, mimicking the phenotype observed in embryos lacking D-Raf. These studies provide the first direct genetic evidence of Rap1-mediated activation of the MAP kinase cascade in eukaryotic organisms.

Identification and characterization of a novel Drosophila melanogaster glutathione S-transferase-containing FLYWCH zinc finger protein.

Glutathione SH-transferase (GST) is a 25-kDa protein and a member of a large family that plays a critical role in the cellular homeostasis of all organisms. In this report, we describe a novel GST-containing protein identified and cloned from Drosophila. This 1045 amino acid protein possesses a zinc finger domain with a tandem array of four FLYWCH zinc finger motifs at its N-terminus and a C-terminal domain that shares a 46% homology with GST. The gene maps to chromosome 3 at position 84C6. Further characterization of this protein shows that it localizes to the cytoplasm of fly cells and is expressed through all stages of fly embryonic development. It binds to glutathione-S agarose beads in vitro. These results indicate that this new protein belongs to the GST family, thus named a Drosophila GST-containing FLYWCH zinc finger protein (dGFZF).

The acetyltransferase activity of CBP is required for wingless activation and H4 acetylation in Drosophila melanogaster.

CBP is a critical coactivator of transcription, but little is understood about the importance of its intrinsic acetyltransferase (AT) activity in gene activation in vivo. We show that the intrinsic AT function of CBP in Drosophila melanogaster (dCBP) is necessary to maintain a dCBP overexpression phenotype in the eye, for the in vivo activation of a specific target gene, wingless, and for the global acetylation of histone H4. These findings indicate that a point mutation which alters the intrinsic AT activity of CBP (only one of many CBP functions) has profound effects on CBP-induced gene activation in a physiologically intact transcription system. Furthermore, the effects of CBP AT activity are not limited to a few specific promoters, but rather CBT AT activity may play a role in regulating global histone acetylation throughout the developing organism.

The interaction between the coactivator dCBP and Modulo, a chromatin-associated factor, affects segmentation and melanotic tumor formation in Drosophila.

The development of Drosophila requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of Drosophila T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. In this report, we provide evidence that dCBP may regulate the formation of chromatin states through interactions with the modulo (mod) gene product, a protein that is thought to be involved in chromatin packaging. We demonstrate that dCBP and Modulo bind in vitro and in vivo, that mutations in mod enhance the embryonic phenotype of a dCBP mutation, and that dCBP mutations enhance the melanotic tumor phenotype characteristic of mod homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure.

A Drosophila homologue of Sir2 modifies position-effect variegation but does not affect life span.

Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability. In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, we identified the Drosophila homolog of a yeast chromatin regulator, Sir2. In yeast, Sir2 silences genes via an intrinsic NAD(+)-dependent histone deacetylase activity. In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans. In this report, we characterize the Drosophila Sir2 (dSir2) gene and its product and describe the generation of dSir2 amorphic alleles. We found that dSir2 expression is developmentally regulated and that dSir2 has an intrinsic NAD(+)-dependent histone deacetylase activity. The dSir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, dSir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function. However, mutations in dSir2 do not shorten life span as predicted from studies in yeast and worms.