PLoS One. 2017 Sep 29;12(9):e0185516. doi: 10.1371/journal.pone.0185516.

Low RNA Polymerase III activity results in up regulation of HXT2 glucose transporter independently of glucose signaling and despite changing environment.

Malgorzata Adamczyk, Roza Szatkowska.

Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland

 

Abstract

Background

Saccharomyces cerevisiae responds to glucose availability in the environment, inducing the expression of the low-affinity transporters and high-affinity transporters in a concentration dependent manner. This cellular decision making is controlled through finely tuned communication between multiple glucose sensing pathways including the Snf1-Mig1, Snf3/Rgt2-Rgt1 (SRR) and cAMP-PKA pathways.

Results

We demonstrate the first evidence that RNA Polymerase III (RNAP III) activity affects the expression of the glucose transporter HXT2 (RNA Polymerase II dependent – RNAP II) at the level of transcription. Down-regulation of RNAP III activity in an rpc128-1007 mutant results in a significant increase in HXT2 mRNA, which is considered to respond only to low extracellular glucose concentrations. HXT2 expression is induced in the mutant regardless of the growth conditions either at high glucose concentration or in the presence of a non-fermentable carbon source such as glycerol. Using chromatin immunoprecipitation (ChIP), we found an increased association of Rgt1 and Tup1 transcription factors with the highly activated HXT2 promoter in the rpc128-1007 strain. Furthermore, by measuring cellular abundance of Mth1 corepressor, we found that in rpc128-1007, HXT2 gene expression was independent from Snf3/Rgt2-Rgt1 (SRR) signaling. The Snf1 protein kinase complex, which needs to be active for the release from glucose repression, also did not appear perturbed in the mutated strain.

Conclusions/Significance

These findings suggest that the general activity of RNAP III can indirectly affect the RNAP II transcriptional machinery on the HXT2 promoter when cellular perception transduced via the major signaling pathways, broadly recognized as on/off switch essential to either positive or negative HXT gene regulation, remain entirely intact. Further, Rgt1/Ssn6-Tup1 complex, which has a dual function in gene transcription as a repressor-activator complex, contributes to HXT2 transcriptional activation.

PMID: 28961268

 

Supplement:

Our recent studies have indicated an unusual relationship between RNA Polymerase III (RNAP III) activity and expression of genes dependent on RNA Polymerase II (RNAP II) encoding proteins such as glucose transporters in yeast. Glucose transporters differ in their expression pattern, which depends on the glucose availability in the extracellular environment. Glucose transporters that have high affinity to the substrate are expressed, when glucose is limited in the medium (at the concentration < 0.1%). Accordingly, low affinity glucose transporters genes are transcribed, when concentration of glucose is at high extracellular level.

Many types of cancer cells express high affinity glucose transporters, even though glucose is abundant in the environment and normal cells do not show this effect under aforementioned conditions.

Hxt2 shows 27% similarity in amino acids transmembrane sequence to GLUT1 transporter sequence (Baldwin 1993). Both proteins are members of the same major facilitator superfamily. High affinity glucose transporter 1 (GLUT1) is overexpressed in breast cancer (Grover-McKay et al. 1998), stage I nonsmall cell lung carcinoma (Younes et al. 1997), ovarian carcinoma (Cantuaria et al. 2001) and cervical tumors (Rudlowski et al. 2003). Moreover, expression of the low concentration glucose transporter 3 (GLUT3) is up-regulated in stage I nonsmall cell lung carcinoma (Younes et al. 1997), prostate cancer (Vaz et al. 2016), brain tumor-initiating cells (Flavahan et al. 2013) and GLUT1 concentration is increased in plasma membrane of human fibroblasts and skeletal muscle of non-insulin-dependent diabetes patients.

Our basic research data on the interplay between glucose signaling transduction and RNA polymerase III activity in yeast facilitates the efforts of scientific community to discover new drug targets for the efficient treatment of cancerous transformation in mammalian cells.

We used two yeast Saccharomyces cerevisiae mutants, maf1-D and rpc128-1007 with respectively, up- and down-regulated RNAP III activity (Ciesla et al. 2007) to examine the regulation of expression of the high-affinity glucose transporter gene HXT2.

Maf1 is a negative regulator of RNAP III (Boguta et al. 1997), whose deficiency results in  elevated tRNA cellular levels. Whereas point  mutation Gly1007Ala in the second largest RNAP III subunit C128 (Ciesla et al. 2007) adversely affects the assembly of the holoenzyme and as a consequence the enzyme’s activity decreases, which is correlated with reduced tRNA synthesis in the mutant cells.

We measured HXT2 transcript levels in maf1-D and rpc128-1007 in rich medium under repression conditions in the presence of high glucose concentration (2%) and non-fermentative carbon source (2% glycerol) (Figure 1). In the case of the wild type cells, high glucose concentration leads to inhibition of HXT2 expression due to “glucose repression” mechanism via Snf1-Mig1 signaling pathway (Ozcan and Johnston, 1995). On the other hand, lack of glucose in the extracellular environment represses HXT2 gene expression via Snf3/Rgt2-Rgt1 (SRR) pathway. Surprisingly, under both conditions, maf1-Δ and rpc128-1007 strains showed elevated HXT2 steady-state transcript levels in comparison to isogenic wild-type strain when measured by RealTime qPCR.

Our primary data suggested that the mutant strains lost an environmental sensitivity for glucose. HXT2 mRNA was increased in rpc128-1007 around 23 fold on 2% glucose medium and 340 fold on glycerol-based medium. We presumed that glucose signaling may be perturbed in the maf1-Δ and in rpc128-1007 mutants, we excluded the possibility for rpc128-1007 mutant providing a few lines of evidence.

To verify, whether the Snf1 kinase dependent “glucose repression” mechanism is present in the mutant strains we measured invertase activity. SUC2 gene, which encoded invertase, is strongly regulated at the transcriptional level by glucose availability in the extracellular environment and Snf1 activity (Ozcan et al. 1998). Low invertase activity detected in rpc128-1007 and maf1-Δ on high glucose was strong evidence that increased HXT2 expression under the aforementioned conditions is not the result of a perturbation of the glucose repression mechanism via the Snf1 pathway.

Additionally, we performed Chromatin Immunoprecipitation (ChIP) assays using Mig1-3HA protein fusion. Constitutive hyperactivation of Snf1 kinase in reg1-Δ results in Mig1 constitutive phosphorylation that prevents the downregulation of HXT2 on high glucose (Ozcan et al. 1995).

However our data indicated, that Mig1 protein, which is a negative regulator of glucose repressed genes, such as HXT2 binds to the HXT2 promoter accordingly to the factual, high glucose availability, but does not represses the gene in mutant cells with low RNAP III activity. Taking together, observation of the phosphorylation status of Mig1 and its DNA binding activity in rpc128-1007compared with the control strain, indicated that glucose signal forwarded via Snf1 downstream to Mig1 could not account for the significant increase in HXT2 expression observed on both carbon sources in cells carrying a mutated C128 RNAP III subunit.

We also found, that Rgt1 transcriptional factor, which recognizes three binding motifs on the HXT2 promoter (Kim et al. 2003, Kaniak et al. 2004, Kim, 2009) had a higher occupancy on the HXT2 promoter in the rpc128-1007 when grown on non-fermentable carbon source. Whereas Tup1 chromatin-silencing transcriptional regulator, occupancy was significantly increased on HXT2 promoter both on fermentable and non-fermentable carbon source.

Rgt1 is the transcriptional factor that receives information regarding the external glucose concentration via Snf3/Rgt2-Rgt1 (SRR) signaling pathway and the cAMP-PKA pathway to regulate HXT expression. Rgt1 bound to DNA interacts with the Ssn6-Tup1 repressor-coactivator complex. When glucose is scarce, Mth1 is responsible for maintaining HXT2 repression by sustaining the interaction between Rgt1 and the Ssn6-Tup1 complex. Mth1 facilities the transcriptional repression of HXT genes by regulating the function of the Rgt1 transcriptional repressor by preventing its phosphorylation by PKA kinase (cAMP-PKA pathway master component) in the nucleus, in the absence of glucose (Kim et al. 2003, Lakshmanan et al., 2012, Kaniak et al. 2004, Polish et al. 2005, Kim and Johnston, 2006, Roy et al. 2013) Keeping the level of co-repressors is crucial to maintain repression. Mth1, via an alteration of its own abundance, mirrors the extracellular glucose concentration. Therefore we measured abundance of Mth1 in the mutant strain with compromised RNAP III. We found that Mth1 was highly abundant in rpc128-1007 and the reference strain during exposure to glycerol and not detectable either in the reference strain nor the mutant on 2% glucose medium. Thus, we concluded that cells with defected RNAP III have an accurate perception of external glucose availability via Snf3/Rgt2-Rgt1 (SRR), however the glucose signaling mechanisms involved in repression and induction of the gene become redundant. Our results¸ the significant enrichment of Tup1 co-repressor on chromatin (human homolog – Groucho protein), regardless carbon source availability, suggest a model, in which Rgt1 with Ssn6-Tup1 function as a transcriptional co‑activator complex rather than a repressor complex, leading to the induction of HXT2 in rpc128-1007. It is likely that intracellular signal, either a protein or a metabolite, serves as a superior regulatory element for Rgt1/Ssn6-Tup1 co-repressor complex, which triggers switching the complex into an activator mode and can wholly account for HXT2 mRNA accumulation in the strain with low RNAP III activity. Since Tup1 interacts with PI(3,5)P2 lipid, and Maf1, the regulator of RNAP III is a target of phosphoinositide 3-kinase (PI3K) signaling that negatively regulates oncogenesis and lipid metabolism in mice (Han and Emr, 2011, Palian et al. 2014), we examined two mutant strains fab1-Δ, that lacks the vacuolar membrane kinase generating PI(3,5)P2 and vac14-Δ, which synthesizes only modest amounts of the metabolite, however our data demonstrated that HXT2 steady state mRNA level, does not seem to be dependent on PI(3,5)P2 intracellular abundance.

In a summary, the Ssn6-Tup1 complex transforms into a co-activator complex due to an unidentified intracellular signal and the expression of HXT2 is induced, which is an exceptional case to the generalized causality model by Zaman, asserting that the cellular perception of nutrient availability establishes the transcriptional pattern in yeast (Zaman et al. 2009).

 

 

Figure 1. Low RNAP III activity caused by Gly1007Ala point mutation in C128 RNAP III subunit, correlates with transcription of HXT2 gene by RNAP II, despite repressing conditions... High concentration of glucose and lack of glucose in the extracellular environment result in repression of HXT2 transcription in the wild type yeast cells. Elevated level of HXT2 mRNA is observed in the rpc128-1007 mutant under both conditions. Rgt2/Snf3-Rgt1 and Snf1-Mig1 signaling pathways are not perturbed in the mutant and the signaling is properly transduced via  Mth1glucose sensor. We propose Ssn6-Tup1 complex as a HXT2 transcription coactivator in rpc128-1007. Under high-glucose conditions, Mth1 degradation occurs. Rgt1, which is phosphorylated by PKA, dissociates from the HXT2 promoter. Mig1, which is bound to the regulatory region, recruits Ssn6-Tup1. The complex transforms into a coactivator complex due to an unidentified intracellular signal and the expression of HXT2 is induced. Under non-fermentable growth conditions in the strain with low RNAP III activity, the Snf3/Rgt2-Rgt1 (SRR) pathway transduces the signal for unfavorable external conditions to Mth1, preventing its degradation. The Rgt1 and Tup1 corepressor complex transforms into an activator complex and strongly induces HXT2 expression.

 

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