Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5' extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5' extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.

Kinetochore inactivation by expression of a repressive mRNA.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.

Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability.

Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.

Identifying protein partners of CLN8, an ER-resident protein involved in neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of neurodegenerative diseases characterized by cognitive and motor decline, epilepsy, visual loss and by lysosomal autofluorescent inclusions. Two distinct clinical phenotypes, the progressive epilepsy with mental retardation (EPMR) and a late-infantile variant of NCLs (CLN8-vLINCL) are associated with mutations in the CLN8 gene that encodes a transmembrane protein predominantly located to the endoplasmic reticulum (ER). To gain insight into the function of CLN8 protein, we employed the split-ubiquitin membrane-based yeast two-hybrid (MYTH) system, which detects protein-protein interactions in a membrane environment, using the full-length human CLN8 as bait and a human brain cDNA library as prey. We identified several potential protein partners of CLN8 and especially referred to VAPA, c14orf1/hERG28, STX8, GATE16, BNIP3 and BNIP3L proteins that are associated with biologically relevant processes such as synthesis and transport of lipids, vesicular/membrane trafficking, autophagy/mitophagy and apoptosis. Interactions of CLN8 with VAPA and GATE16 were further validated by co-immunoprecipitation and co-localization assays in mammalian cells. Using a new C-terminal-oriented CLN8 antibody, CLN8-VAPA interaction was also confirmed by co-staining in close spatial proximity within different CNS tissues. The results of this study shed light on potential interactome networks of CLN8 and provide a powerful starting point for understanding protein function(s) and molecular aspects of diseases associated with CLN8 deficiency.

ATAC-king the complexity of SAGA during evolution.

The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) coactivator complex exerts functions in gene expression, including activator interaction, histone acetylation, histone deubiquitination, mRNA export, chromatin recognition, and regulation of the basal transcription machinery. These diverse functions involve distinct modules within this multiprotein complex. It has now become clear that yeast SAGA has diverged during metazoan evolution into two related complexes, SAGA and ATAC, which exist in two flavors in vertebrates. The compositions of metazoan ATAC and SAGA complexes have been characterized, and functional analyses indicate that these complexes have important but distinct roles in transcription, histone modification, signaling pathways, and cell cycle regulation.

Tight cooperation between Mot1p and NC2ß in regulating genome-wide transcription, repression of transcription following heat shock induction and genetic interaction with SAGA.

TATA-binding protein (TBP) is central to the regulation of eukaryotic transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1p or the NC2 complex. Recent evidence suggests that Mot1p, NC2 and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1p and NC2ßduring basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes. Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2ß depletion during heat shock resulted in failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2ß displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results support the model that Mot1p and NC2ß directly cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress.

The structural plasticity of SCA7 domains defines their differential nucleosome-binding properties.

SAGA (Spt-Ada-Gcn5 acetyltransferase), a coactivator complex involved in chromatin remodelling, harbours both histone acetylation and deubiquitination activities. ATXN7/Sgf73 and ATXN7L3, two subunits of the SAGA deubiquitination module, contain an SCA7 domain characterized by an atypical zinc-finger. We show that the yeast Sgf73-SCA7 domain is not required to recruit Sgf73 into SAGA. Instead, it binds to nucleosomes, a property that is conserved in the human ATXN7-SCA7 domain but is lost in the ATXN7L3 domain. The solution structures of the SCA7 domain of both ATXN7 and ATXN7L3 reveal a new, common zinc-finger motif at the heart of two distinct folds, providing a molecular basis for the observed functional differences.

Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex.

The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.

In-depth profiling of post-translational modifications on the related transcription factor complexes TFIID and SAGA.

The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here, we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multipronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate <1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally, for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach, we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive prefractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.