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.

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for spreading of repressive chromatin. The Su(Hw) protein is responsible for activity of the best-studied Drosophila insulators. Here we demonstrate that an evolutionarily conserved protein, E(y)2/Sus1, is recruited to the Su(Hw) insulators via binding to the zinc-finger domain of Su(Hw). Partial inactivation of E(y)2 in a weak mutation, e(y)2(u1), impairs only the barrier, but not the enhancer-blocking, activity of the Su(Hw) insulators. Whereas neither su(Hw)(-) nor e(y)2(u1) affects fly viability, their combination proves lethal, testifying to functional interaction between Su(Hw) and E(y)2 in vivo. Apparently, different domains of Su(Hw) recruit proteins responsible for enhancer-blocking and for the barrier activity.

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...

Enhancer-mediated promoter activation is a fundamental mechanism of gene regulation in eukaryotes (10, 44). Recently, sequences in different organisms have been identified that constrain enhancer action. These elements, known as insulators, block communication between an enhancer and promoter only when the insulator is positioned between these regulatory elements. Similarly, insulators prevent silencer interactions with promoters (6,10,30,44,45). The properties of insulators are exemplified by the gypsy insulator that originally was found in the gypsy transposable element (19,24).Genetic and molecular approaches have led to the identification and characterization of three proteins, Suppressor of Hairy wing [Su(Hw)], Mod(mdg4)-67.2, and CP190, that are required for the activity of the gypsy insulator (6, 36). Su(Hw) is a zinc finger protein that binds 12 directly repeated copies of a short sequence motif in the gypsy insulator (9, 42). In addition, Su(Hw) has two acidic domains located at the amino (N) and carboxyl (C) termini of the protein and a C-terminal enhancer-blocking region that is essential for insulation (23, 29). The mod(mdg4) gene, also known as E(var)3-93D, encodes a large set of protein isoforms with specific functions in regulating the chromatin structure of different genes (3). All isoforms encoded by mod(mdg4) contain a BTB/POZ domain and a glutamine-rich (Q) region in the N terminus (3, 7). The BTB (broad complex, tramtrack, bric-a-brac) or POZ (poxvirus and zinc finger) domain identifies a large family of proteins in organisms from yeast to humans (43,47). This domain functions as a protein interaction domain that facilitates homodimer (2, 33, 34) and heterodimer formation as well as oligomerization (11,28,37). One of the mod(mdg4)-encoded protein isoforms, Mod(mdg4)-67.2, interacts with the enhancer-blocking domain of the Su(Hw) protein (12, 20) through a C-terminal acidic domain. This domain is affected in two viable mutations mod(mdg4) u1 and mod(mdg4) T6 (12, 15). The third component of the insulator complex, CP190, also contains a BTB domain (38). It was suggested that Mod(mdg4)-67.2 and CP190 interact through their BTB domains.The mod(mdg4) u1 and mod(mdg4) T6 mutations have varying effects on insulator function, resulting in partial restoration of enhancer-promoter communication in some cases, while transforming the insulator into a silencer in others (4,12,13,14,15,22). The domains of the Mod(mdg4)-67.2 protein required for the insulator and antirepression activity are not determined. Although the essential role of the BTB domain for Mod-(mdg4)-67.2 activity was predicted in the previous studies, this postulate has not been proven (12,20). Here we examined the role of the BTB domain in the functional activities of the Mod(mdg4)-67.2 protein.The structure of the BTB domain has been examined for mammalian transcriptional repressors 34). The high degree of sequence identity between the BTB domains of Mod(mdg4), Bcl-6, and PZLF (1) allowed us to predict key residues of the Mod(mdg4)-67.2 ...

The presence of general transcription factors and other coactivators at the Drosophila hsp70 gene promoter in vivo has been examined by polytene chromosome immunofluorescence and chromatin immunoprecipitation at endogenous heat-shock loci or at a hsp70 promoter-containing transgene. These studies indicate that the hsp70 promoter is already occupied by TATA-binding protein (TBP) and several TBP-associated factors (TAFs), TFIIB, TFIIF (RAP30), TFIIH (XPB), TBP-free͞TAF-containg complex (GCN5 and TRRAP), and the Mediator complex subunit 13 before heat shock. After heat shock, there is a significant recruitment of the heatshock transcription factor, RNA polymerase II, XPD, GCN5, TRRAP, or Mediator complex 13 to the hsp70 promoter. Surprisingly, upon heat shock, there is a marked diminution in the occupancy of TBP, six different TAFs, TFIIB, and TFIIF, whereas there is no change in the occupancy of these factors at ecdysone-induced loci under the same conditions. Hence, these findings reveal a distinct mechanism of transcriptional induction at the hsp70 promoters, and further indicate that the apparent promoter occupancy of the general transcriptional factors does not necessarily reflect the transcriptional state of a gene.polytene chromosomes ͉ TATA-binding protein, ͉ TATA-binding protein-associated factors ͉ TFIID ͉ transcription T ranscription initiation of protein-coding genes by RNA polymerase II (Pol II) involves the polymerase along with the general transcription initiation factors (GTFs), which include TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1). TFIID, which comprises TATA box-binding protein (TBP) and TBPassociated factors (TAFs), recognizes the core promoter in a sequence-specific manner (2, 3). TAFs are also present in other transcription-related multiprotein complexes, such as TBP-free͞ TAF-containing complex (TFTC), STAGA, and PCAF͞GCN5 (4-7). These complexes are homologous to yeast SAGA complexes and contain GCN5 histone acetyltransferase, TRRAP protein, and several TAFs (5). In addition, other transcriptional coactivators, such as Mediator complexes, promote regulatory interactions between transcriptional activators and the GTFs (refs. 8-10 and references therein).The hsp70 gene cluster has served as an important focus for the analysis of transcriptional activation upon heat shock in Drosophila melanogaster. The region upstream of the hsp70 TATA element contains multiple binding sites for the sequence-specific regulatory proteins GAGA factor (GAF) and heat-shock factor (HSF). Before heat shock, GAF has been observed to reside on the hsp70 promoter (11). The binding of GAF appears to maintain the promoter region in a nucleosome-free conformation (12-16). The existence of an open chromatin conformation of the hsp70 promoter in noninduced conditions has been thought to allow access of the GTFs to the core promoter for rapid transcriptional induction.The GTFs form a transcription complex wherein the polymerase initiates transcription and then pauses Ϸ17-37 nt downstream of the start site (17). Then, upon he...