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.

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...

Metazoan E(y)2/ENY2 is a multifunctional protein important for transcription activation and mRNA export, being a component of SAGA/TFTC and the mRNA export complex AMEX. Here, we show that ENY2 in Drosophila is also stably associated with THO, the complex involved in mRNP biogenesis. The ENY2-THO complex is required for normal Drosophila development, functioning independently on SAGA and AMEX. ENY2 and THO arrive on the transcribed region of the hsp70 gene after its activation, and ENY2 plays an important role in THO recruitment. ENY2 and THO show no direct association with elongating RNA polymerase II. Recruitment of ENY2 and THO occurs by their loading onto nascent mRNA, apparently immediately after its synthesis, while the AMEX component Xmas-2 is loaded onto mRNA at a later stage. Knockdown of either ENY2 or THO, but not SAGA or AMEX, affects the processing of the transcript's 39 end. Thus, ENY2, as a shared subunit of several protein complexes governing the sequential steps of gene expression, plays an important role in the coordination of these steps.[Keywords: THO; mRNA export; mRNP formation; gene expression; protein complex; ENY2] Supplemental material is available at http://www.genesdev.org.

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.