Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1.

Transcriptional corepressors Sin3, Cyc8 and Tup1 are important for downregulation of gene expression by recruiting various histone deacetylases once they gain access to defined genomic locations by interaction with pathway-specific repressor proteins. In this work we systematically investigated whether 17 yeast repressor proteins (Cti6, Dal80, Fkh1, Gal80, Mig1, Mot3, Nrg1, Opi1, Rdr1, Rox1, Sko1, Ume6, Ure2, Xbp1, Yhp1, Yox1 and Whi5) representing several unrelated regulatory pathways are able to bind to Sin3, Cyc8 and Tup1. Our results show that paired amphipathic helices 1 and 2 (PAH1 and PAH2) of Sin3 are functionally redundant for some regulatory pathways. WD40 domains of Tup1 proved to be sufficient for interaction with repressor proteins. Using length variants of selected repressors, we mapped corepressor interaction domains (CIDs) in vitro and assayed gene repression in vivo. Systematic comparison of CID minimal sequences allowed us to define several related positional patterns of hydrophobic amino acids some of which could be confirmed as functionally supported by site-directed mutagenesis. Although structural predictions indicated that certain CIDs may be α-helical, most repression domains appear to be randomly structured and must be considered as intrinsically disordered regions (IDR) adopting a defined conformation only by interaction with a corepressor.

Differential modulation of Target of Rapamycin activity under single and combined iron and sulfur deficiency in tomato plants.

Over the past few decades, a close relationship between sulfur (S) and iron (Fe) in terms of functionality and nutrition was demonstrated in the tomato. However, very little is known about the regulatory mechanisms underlying S/Fe interactions. Recently, the potential role of citrate in plant adaptation to Fe deficiency and combined S and Fe deficiency has been described. It is known that an impaired organic acid metabolism may stimulate a retrograde signal, which has been proven to be linked to the Target of Rapamycin (TOR) signaling in yeast and animal cells. Recent reports provided evidence of TOR involvement in S nutrient sensing in plants. This suggestion prompted us to investigate whether TOR may play a role in the cross-talk of signaling pathway occurring during plant adaptation to combined nutrient deficiency of Fe and S. Our results revealed that Fe deficiency elicited an increase of TOR activity associated with enhanced accumulation of citrate. In contrast, S deficiency resulted in decreased TOR activity and citrate accumulation. Interestingly, citrate accumulated in shoots of plants exposed to combined S/Fe deficiency to values between those found in Fe- and S-deficient plants, again correlated with TOR activity level. Our results suggest that citrate might be involved in establishing a link between plant response to combined S/Fe deficiency and the TOR network.

The plant TOR kinase tunes autophagy and meristem activity for nutrient stress-induced developmental plasticity.

Plants, unlike animals, respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. Here we discovered that antagonistic activation of the Target of Rapamycin (TOR) kinase in Arabidopsis thaliana roots and shoots is essential for the nutrient deprivation-induced increase in the root-to-shoot ratio to improve foraging for mineral ions. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy, resulting in enhanced carbon allocation to the root. The allocation of carbon to the roots is facilitated by the specific upregulation of the sucrose-transporter genes SWEET11/12 in shoots. SWEET11/12 activation is indispensable for enabling sucrose to act as a carbon source for growth and as a signal for tuning root apical meristem activity via glucose-TOR signaling. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth to enhance mining for new sulfate resources in the soil. We provide direct evidence that the organ-specific regulation of autophagy is essential for the increased root-to-shoot ratio in response to sulfur limitation. These findings uncover how sulfur limitation controls the central sensor kinase TOR to enable nutrient recycling for stress-induced morphological adaptation of the plant body.

Transcriptional repressor Gal80 recruits corepressor complex Cyc8-Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon.

Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA-Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1-lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

Micrografting Provides Evidence for Systemic Regulation of Sulfur Metabolism between Shoot and Root.

The uptake of sulfate by roots and its reductive assimilation mainly in the leaves are not only essential for plant growth and development but also for defense responses against biotic and abiotic stresses. The latter functions result in stimulus-induced fluctuations of sulfur demand at the cellular level. However, the maintenance and acclimation of sulfur homeostasis at local and systemic levels is not fully understood. Previous research mostly focused on signaling in response to external sulfate supply to roots. Here we apply micrografting of Arabidopsis wildtype knock-down sir1-1 mutant plants that suffer from an internally lowered reductive sulfur assimilation and a concomitant slow growth phenotype. Homografts of wildtype and sir1-1 confirm the hallmarks of non-grafted sir1-1 mutants, displaying substantial induction of sulfate transporter genes in roots and sulfate accumulation in shoots. Heterografts of wildtype scions and sir1-1 rootstocks and vice versa, respectively, demonstrate a dominant role of the shoot over the root with respect to sulfur-related gene expression, sulfate accumulation and organic sulfur metabolites, including the regulatory compound O-acetylserine. The results provide evidence for demand-driven control of the shoot over the sulfate uptake system of roots under sulfur-sufficient conditions, allowing sulfur uptake and transport to the shoot for dynamic responses.

Forkhead transcription factor Fkh1: insights into functional regulatory domains crucial for recruitment of Sin3 histone deacetylase complex.

Transcription factors are inextricably linked with histone deacetylases leading to compact chromatin. The Forkhead transcription factor Fkh1 is mainly a negative transcriptional regulator which affects cell cycle control, silencing of mating-type cassettes and induction of pseudohyphal growth in the yeast Saccharomyces cerevisiae. Markedly, Fkh1 impinges chromatin architecture by recruiting large regulatory complexes. Implication of Fkh1 with transcriptional corepressor complexes remains largely unexplored. In this work we show that Fkh1 directly recruits corepressors Sin3 and Tup1 (but not Cyc8), providing evidence for its influence on epigenetic regulation. We also identified the specific domain of Fkh1 mediating Sin3 recruitment and substantiated that amino acids 51-125 of Fkh1 bind PAH2 of Sin3. Importantly, this part of Fkh1 overlaps with its Forkhead-associated domain (FHA). To analyse this domain in more detail, selected amino acids were replaced by alanine, revealing that hydrophobic amino acids L74 and I78 are important for Fkh1-Sin3 binding. In addition, we could prove Fkh1 recruitment to promoters of cell cycle genes CLB2 and SWI5. Notably, Sin3 is also recruited to these promoters but only in the presence of functional Fkh1. Our results disclose that recruitment of Sin3 to Fkh1 requires precisely positioned Fkh1/Sin3 binding sites which provide an extended view on the genetic control of cell cycle genes CLB2 and SWI5 and the mechanism of transcriptional repression by modulation of chromatin architecture at the G2/M transition.

Functional analysis of Cti6 core domain responsible for recruitment of epigenetic regulators Sin3, Cyc8 and Tup1.

Mapping of effective protein domains is a demanding stride to disclose the functional relationship between regulatory complexes. Domain analysis of protein interactions is requisite for understanding the pleiotropic responses of the respective partners. Cti6 is a multifunctional regulator for which we could show recruitment of co-repressors Sin3, Cyc8 and Tup1. However, the responsible core domain tethering Cti6 to these co-repressors is poorly understood. Here, we report the pivotal domain of Cti6 that is indispensable for co-repressor recruitment. We substantiated that amino acids 450-506 of Cti6 bind PAH2 of Sin3. To analyse this Cti6-Sin3 Interaction Domain (CSID) in more detail, selected amino acids within CSID were replaced by alanine. It is revealed that hydrophobic amino acids V467, L481 and L491 L492 L493 are important for Cti6-Sin3 binding. In addition to PAH2 of Sin3, CSID also binds to tetratricopeptide repeats (TPR) of Cyc8. Indeed, we could demonstrate Cti6 recruitment to promoters of genes, such as RNR3 and SMF3, containing iron-responsive elements (IRE). Importantly, Sin3 is also recruited to these promoters but only in the presence of functional Cti6. Our findings provide novel insights toward the critical interaction domain in the co-regulator Cti6, which is a component of regulatory complexes that are closely related to chromatin architecture and the epigenetic status of genes that are regulated by pleiotropic co-repressors.

Correction to: Promoter recruitment of corepressors Sin3 and Cyc8 by activator proteins of the yeast Saccharomyces cerevisiae.

The original version of this article unfortunately contained a mistake.

Promoter recruitment of corepressors Sin3 and Cyc8 by activator proteins of the yeast Saccharomyces cerevisiae.

It is generally assumed that pathway-specific transcriptional activators recruit pleiotropic coactivators (such as chromatin-modifying complexes or general transcription factors), while specific repressors contact pleiotropic corepressors creating an inaccessible chromatin by the action of histone deacetylases. We have previously shown that the negative regulator Opi1 of yeast phospholipid biosynthesis inhibits transcription by recruiting corepressors Sin3 and Cyc8 in the presence of precursor molecules inositol and choline. To get access to its target genes, Opi1 physically contacts and counteracts DNA-bound activator Ino2. By using chromatin immunoprecipitation, we show that Sin3 and Cyc8 can be detected at Opi1 target promoters INO1 and CHO2 under repressing and derepressing conditions and that corepressor binding is effective even in the absence of Opi1, while Ino2 is absolutely required. Thus, corepressors may be recruited not only by repressors but also by activators such as Ino2. Indeed, we could demonstrate direct interaction of Ino2 with Sin3 and Cyc8. The Opi1 repressor interaction domain within Ino2 is also able to contact Sin3 and Cyc8. Recruitment of corepressors by an activator is not a regulatory exception as we could show that activators Pho4 and Hac1 also contain domains being able to interact with Sin3 and Cyc8.