Chi Kwan Tsang

Nutrient regulates Tor1 nuclear localization and association with rDNA promoter

TOR is the target of the immunosuppressant rapamycin and a key regulator of cell growth. It modulates diverse cellular processes in the cytoplasm and nucleus, including the expression of amino acid transporters, ribosomal RNAs and ribosomal proteins. Despite considerable recent progress, little is known about the spatial and temporal regulation of TOR signalling, particularly that leading into the nucleus. Here we show that Tor1 is dynamically distributed in the cytoplasm and nucleus in yeast. Tor1 nuclear localization is nutrient dependent and rapamycin sensitive: starvation or treatment with rapamycin causes Tor1 to exit from the nucleus. Tor1 nuclear localization is critical for 35S rRNA synthesis, but not for the expression of amino acid transporters and ribosomal protein genes. We show further that Tor1 is associated with 35S ribosomal DNA (rDNA) promoter chromatin in a rapamycin- and starvation-sensitive manner; this association is necessary for 35S rRNA synthesis and cell growth. These results indicate that the spatial regulation of TOR complex 1 (TORC1) might be involved in differential control of its target genes. TOR is known as a classic cytoplasmic kinase that mediates the cytoplasm-to-nucleus signalling by controlling the localization of transcription factors. Our data indicate that TOR might be more intimately involved in gene regulation than previously thought.

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3–Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site‐specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Ü and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin‐mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Regulation of Subtelomeric Silencing during Stress Response

Sir proteins play a critical role in silent chromatin domains. While mutations can cause derepression of heterochromatin, it remains unclear whether silencing is actively involved in transcriptional control under changing environmental conditions. We find that TOR inhibits Sir3 phosphorylation. Rapamycin or stress induced by chlorpromazine leads to activation of MAP kinase Mpk1/Slt2, which phosphorylates Sir3. Sir3 hyperphosphorylation is correlated with reduced subtelomeric silencing, increased subtelomeric cell wall gene expression, and stress resistance to chlorpromazine, but does not affect the silent HML and rDNA loci. Based on these observations, we propose that regulation of silencing may be used to control gene expression at specific silent chromatin domains in response to stress and possibly other environmental changes.

Mechanisms of regulation of RNA polymerase III‐dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III‐transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1‐v allele. In W303 cells that carry the SSD1‐d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm‐to‐nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm‐to‐nucleolus translocation. Together, these observations provide new insights into the chromatin‐dependent mechanism by which TORC1 controls transcription by Pol III.

Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance

Summary Superoxide dismutase 1 (Sod1) has been known for nearly half a century for catalysis of superoxide to hydrogen peroxide. Here we report a new Sod1 function in oxidative signaling: in response to elevated endogenous and exogenous reactive oxygen species (ROS), Sod1 rapidly relocates into the nucleus, which is important for maintaining genomic stability. Interestingly, H2O2 is sufficient to promote Sod1 nuclear localization, indicating that it is responding to general ROS rather than Sod1 substrate superoxide. ROS signaling is mediated by Mec1/ATM and its effector Dun1/Cds1 kinase, through Dun1 interaction with Sod1 and regulation of Sod1 by phosphorylation at S60, 99. In the nucleus, Sod1 binds to the promoters and regulates the expression of oxidative resistance and repair genes. Altogether, our study unravels an unorthodox function of Sod1 as a transcription factor and elucidates the regulatory mechanism for its localization.

Nutrient starvation promotes condensin loading to maintain rDNA stability.

Nutrient starvation or rapamycin treatment, through inhibition of target of rapamycin, causes condensation of ribosomal DNA (rDNA) array and nucleolar contraction in budding yeast. Here we report that under such conditions, condensin is rapidly relocated into the nucleolus and loaded to rDNA tandem repeats, which is required for rDNA condensation. Rpd3‐dependent histone deacetylation is necessary and sufficient for condensins relocalization and loading to rDNA array, suggesting that histone modification plays a regulatory role for condensin targeting. Rapamycin independently, yet coordinately, inhibits rDNA transcription and promotes condensin loading to rDNA array. Unexpectedly, we found that inhibition of rDNA transcription in the absence of condensin loading leads to rDNA instability. Our data suggest that enrichment of condensin prevents rDNA instability during nutrient starvation. Together, these observations unravel a novel role for condensin in the maintenance of regional genomic stability.

Targeting mTOR as a novel therapeutic strategy for traumatic CNS injuries

The adult central nervous system (CNS) has a remarkable ability to repair itself. However, severe brain and spinal cord injuries (SCIs) cause lasting disability and there are only a few therapies that can prevent or restore function in such cases. In this review, we provide an overview of traumatic CNS injuries and discuss several emerging pharmacological options that have shown promise in preclinical and early clinical studies. We highlight therapies that modulate mammalian target of rapamycin (mTOR) signaling, a pathway that is well known for its roles in cell growth, metabolism and cancer. Interestingly, this pathway is also gaining newfound attention for its role in CNS repair and regeneration.

Compacting DNA During the Interphase: Condensin Maintains rDNA Integrity

During mitosis, condensin is responsible for folding chromatin fibers into highly compact chromosomes, ensuring the faithful segregation of replicated chromosomes into daughter cells after each cell division. Our laboratory has unexpectedly found that condensin is capable of compacting DNA during the interphase: upon nutrient starvation, condensin is loaded to the rDNA array, leading to DNA condensation in this region. This subchromosomal DNA condensation appears to protect the integrity of the rDNA array. These observations provide the first microscopic evidence of DNA compaction by condensin outside mitosis. In addition, they show that condensin is also highly regulated during the interphase.

MAF1 suppresses AKT‐mTOR signaling and liver cancer through activation of PTEN transcription

The phosphatidylinositol 3‐kinase/phosphatidylinositol 3,4,5‐trisphosphate 3‐phosphatase/protein kinase B/mammalian target of rapamycin (PI3K‐PTEN‐AKT‐mTOR) pathway is a central controller of cell growth and a key driver for human cancer. MAF1 is an mTOR downstream effector and transcriptional repressor of ribosomal and transfer RNA genes. MAF1 expression is markedly reduced in hepatocellular carcinomas, which is correlated with disease progression and poor prognosis. Consistently, MAF1 displays tumor‐suppressor activity toward in vitro and in vivo cancer models. Surprisingly, blocking the synthesis of ribosomal and transfer RNAs is insufficient to account for MAF1s tumor‐suppressor function. Instead, MAF1 down‐regulation paradoxically leads to activation of AKT‐mTOR signaling, which is mediated by decreased PTEN expression. MAF1 binds to the PTEN promoter, enhancing PTEN promoter acetylation and activity. Conclusion: In contrast to its canonical function as a transcriptional repressor, MAF1 can also act as a transcriptional activator for PTEN, which is important for MAF1s tumor‐suppressor function. These results have implications in disease staging, prognostic prediction, and AKT‐mTOR‐targeted therapy in liver cancer. (Hepatology 2016;63:1928‐1942)