Yulii V. Shidlovskii

SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC

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/TFTC‐type 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 coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

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.

A novel multidomain transcription coactivator SAYP can also repress transcription in heterochromatin

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.

SAYP and Brahma are important for ‘repressive’ and ‘transient’ Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a ‘nucleosomal barrier’ (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both ‘repressive’ and ‘transient’ Pol II pausing.

The DUBm subunit Sgf11 is required for mRNA export and interacts with Cbp80 in Drosophila

SAGA/TFTC is a histone acetyltransferase complex that has a second enzymatic activity because of the presence of a deubiquitination module (DUBm). Drosophila DUBm consists of Sgf11, ENY2 and Nonstop proteins. We show that Sgf11 has other DUBm-independent functions. It associates with Cbp80 component of the cap-binding complex and is thereby recruited onto growing messenger ribonucleic acid (mRNA); it also interacts with the AMEX mRNA export complex and is essential for hsp70 mRNA export, as well as for general mRNA export from the nucleus. Thus, Sgf11 functions as a component of both SAGA DUBm and the mRNA biogenesis machinery.

The novel regulator of metazoan development SAYP organizes a nuclear coactivator supercomplex.

SAYP is a dual-function transcription coactivator of RNA polymerase II. It is a metazoan-specific factor with regulated expression that is apparently involved in signaling pathways controlling normal development. In Drosophila, SAYP is maternally loaded into the embryo, participates in cell cycle synchronization in early syncytial embryos, and is indispensible for early embryogenesis. SAYP is abundant in many embryonic tissues and imaginal discs in larvae and is crucial for oogenesis in adults. PHF10 is a mammalian homologue of SAYP whose expression is confined to certain tissues in adults. The molecular mechanism of the SAYP function is related to the conserved domain SAY, which assembles a nuclear supercomplex BTFly consisting of Brahma and TFIID coactivators. We suggest that nuclear supercomplexes may be important means of gene-specific regulation of transcription during development.

Transcription co-activator SAYP mediates the action of STAT activator

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome.

ENY2: couple, triple...more?

ENY2/Sus1, a protein involved in the coupling of transcription with mRNA export, is a component of SAGA/TFTC and TREX-2/AMEX complexes. Recently, we have described the association of ENY2 with the third protein complex, THO. Moreover, our data indicate that ENY2 is also associated with other factors, both in the nucleus and cytoplasm. Thus, being a shared components of several protein complexes, ENY2 appears to function as an adapter molecule involved in integration of cellular processes, in particular, subsequent stages of gene expression.

The GAGA factor regulatory network: Identification of GAGA factor associated proteins

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.