SAGA/TFTC-type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTCtype histone acetyl transferase complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. E(y)2 forms also a complex with X-linked male sterile 2 (Xmas-2) to regulate mRNA transport both in normal conditions and after heat shock. Importantly, E(y)2 and Xmas-2 knockdown decreases the contact between the heat-shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation and interferes with transcription. Thus, E(y)2 and Xmas-2 together with SAGA/TFTC function in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.Transcription in eukaryotes is a tightly regulated, multistep process. General transcription factors, gene specific transcriptional activators, and several different cofactors are necessary to access specific loci in the context of eukaryotic chromatin to allow precise initiation of RNA polymerase II (Pol II) transcription. One of the most appealing questions in eukaryotic transcription is how activators transmit their signals to the general transcription machinery to stimulate transcription.Posttranslational modifications of nucleosomal histones have been correlated with the function of chromatin in transcription activation or repression (18, 34). One of the most extensively studied modifications is the acetylation of the highly conserved amino-terminal histone tails. The steady-state level of acetylation of histone proteins is accomplished by the action of histone acetyltransferases (HATs) and histone deacetylases (9, 37). Acetylation affects higher-order folding of chromatin fibers and histone-nonhistone protein interactions (31, 32). Thus, it can increase the affinity of transcription factors for nucleosomal DNA (40,61).A large number of recent studies have provided a direct molecular link between histone acetylation and transcriptional activation (reviewed in references 9 and 30). In these reports, it has been shown that several previously identified coactivators and adaptors of transcription possess i...

The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes.To initiate transcription, each eukaryotic RNA polymerase requires a set of general transcription factors. TFIID, composed of the TATA box-binding protein (TBP) and TBP-associated factors (TAFs), recognizes the core promoter in a sequence-specific manner and is thought to be the only sequence-specific factor that operates with RNA polymerase II (4, 51). The C-terminal core domain of TBP is highly conserved among eukaryotes and contains two symmetrical repeats that fold into a saddle-like structure essential for interaction with the promoter sequences (24,25).A second gene encoding a protein with high homology to the core domain of TBP, TBP-like factor (TLF; also called TRF2 or TLP), was detected in metazoan species (11,23,30,34,38,39,40,41,52). Like TBP, most members of the TLF family have a bipartite structure with a variable N-terminal domain and the highly conserved C-terminal core domain containing two direct repeats (11). TLF was shown to mediate polymerase II transcription initiation and to interact with TFIIA and TFIIB to form a preinitiation complex. However, TLF does not bind to the classical TATA box elements and has been shown to control a set of genes different from those controlled by TBP (12,34,40,41,45,50).Sequence comparison of core domains in the TLF family reveals that they are less conserved in evolution (40 to 45% identity among the metazoan species) than the TBP core domains (about 80% identity between yeast and humans). Thus, while the role of TBP is similar in different species, the function of TLF may have evolved into different regulatory pathways in evolutionarily distant species (11). Studies on the physiological function of TLF in Caenorhabditis elegans, Xenopus laevis, and Danio rerio have demonstrated that TLF is essenti...

Weak hypomorph mutations in the enhancer of yellow genes, e(y)1 and e(y)2, of Drosophila melanogaster were discovered during the search for genes involved in the organization of interaction between enhancers and promoters. Previously, the e(y)1 gene was cloned and found to encode TAF II 40 protein. Here we cloned the e(y)2 gene and demonstrated that it encoded a new ubiquitous evolutionarily conserved transcription factor. The e(y)2 gene is located at 10C3 (36.67) region and is expressed at all stages of Drosophila development. It encodes a 101-amino-acid protein, e(y)2. Vertebrates, insects, protozoa, and plants have proteins which demonstrate a high degree of homology to e(y)2. The e(y)2 protein is localized exclusively to the nuclei and is associated with numerous sites along the entire length of the salivary gland polytene chromosomes. Both genetic and biochemical experiments demonstrate an interaction between e(y)2 and TAF II 40, while immunoprecipitation studies demonstrate that the major complex, including both proteins, appears to be distinct from TFIID. Furthermore, we provide genetic evidence suggesting that the carboxy terminus of dTAF II 40 is important for mediating this interaction. Finally, using an in vitro transcription system, we demonstrate that recombinant e(y)2 is able to enhance transactivation by GAL4-VP16 on chromatin but not on naked DNA templates, suggesting that this novel protein is involved in the regulation of transcription.Despite the enormous progress made in unraveling the complexities of regulated gene transcription during the past few years (9, 21, 30), novel regulatory factors are still being discovered. We are interested in factors that are involved in the organization of interaction between enhancers and promoters, a key process in transcription control. Previously, during the search for such factors, we identified mutations in three genes named enhancers of yellow [e(y)1, e(y)2, and e(y)3], as they influenced yellow expression in the bristles that was activated by a tissue-specific enhancer (15). In combination with the zeste null allele, mutations in these genes strongly inhibited enhancer-dependent white expression (14). The zeste protein recognizes DNA sequences located in the enhancer and promoter regions of certain genes (e.g., the white gene) and is able to mediate protein-protein interactions to generate multimeric zeste complexes (4, 29). In spite of the fact that some mutations of zeste changing the specificity of zeste protein-protein interaction may strongly inhibit the target gene transcription depending on enhancer activity, the null allele of zeste induces only a weak effect on gene expression. The synergistic effects of the zeste null mutation and mutations in the e(y) genes on inhibition of enhancer-dependent white expression suggests that these genes share similar functions.The e(y)1 gene was recently cloned and shown to encode Drosophila melanogaster (d) TAF II 40 protein (also called dTAF II 42) (37). TAF II s or TATA-binding protein-associated factors ar...