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.

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation.coactivators ͉ protein complex A ctivation of transcription by eukaryotic RNA polymerase II (Pol II) requires different groups of coactivators (for reviews, see refs. 1 and 2). The primary function of coactivators is to remodel and modify the chromatin template. Thus, chromatin remodelers of the Brahma (SWI/SNF-related) family play a genome-wide role in activation of Pol II-transcribed genes (3, 4). One more function of coactivators is to further recruit general transcription factors (GTFs) to form the Pol II preinitiation complex. The TFIID coactivator performs this function for most of Pol II-dependent genes (5, 6).Different coactivators recruited to the promoter assist each other and interact in a highly organized gene-specific manner (for a review, see ref. 7). However, this important regulatory step is still poorly understood. The best studied model is that of successive one-by-one recruitment of coactivators, which, in particular, is confirmed by the fact that the recruitment of chromatin-remodeling complexes is usually a prerequisite for the efficient recruitment of GTFs to the promoter (8, 9). The opposite model proposes one-time recruitment of preexisting supercomplex of several coactivators (10-12), although the composition of such supercomplexes described to date appears to be either ambiguous or incomplete.We have described the coactivator SAYP in Drosophila (13). SAYP is present at numerous sites on polytene chromosomes and colocalizes with Pol II in transcriptionally active euchromatin. SAYP homologs in various metazoans have an evolutionarily conserved core containing the SAY domain, which is involved in transcription activation, and 2 PHD fingers (13). Recently, SAYP was found to be associated with the chromatinremodeling Brahma complex of the PBAP subfamily (14). Here, we show that SAYP interacts both with Brahma and with TFIID, assembling them into a stable supercomplex named BTFly (Brahma and TFIID in one assembly). The presence of all BTFly components is crucial for its function in transcription activation. An important fact is that highly purified BTFly contains the full set of TFIID and Brahma subunits and, t...

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.

Enhancers of yellow (e(y)) is a group of genetically and functionally related genes for proteins involved in transcriptional regulation. The e(y)3 gene of Drosophila considered here encodes a ubiquitous nuclear protein that has homologues in other metazoan species. The protein encoded by e(y)3, named Supporter of Activation of Yellow Protein (SAYP), contains an AT-hook, two PHD fingers, and a novel evolutionarily conserved domain with a transcriptional coactivator function. Mutants expressing a truncated SAYP devoid of the conserved domain die at a midembryonic stage, which suggests a crucial part for SAYP during early development. SAYP binds to numerous sites of transcriptionally active euchromatin on polytene chromosomes and coactivates transcription of euchromatin genes. Unexpectedly, SAYP is also abundant in the heterochromatin regions of the fourth chromosome and in the chromocenter, and represses the transcription of euchromatin genes translocated to heterochromatin; its PHD fingers are essential to heterochromatic silencing. Thus, SAYP plays a dual role in transcription regulation in euchromatic and heterochromatic regions.

The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.