Ronald Nicholas Laribee

Chromatin is dynamically regulated in response to nutrient flux to promote the transcriptional changes needed for adaptation. The mechanistic target of rapamycin complex 1 (yeast TORC1) kinase integrates nutrient signaling with chromatin regulation, yet whether chromatin signals back to regulate TORC1 remains unclear. We find that a histone H3 lysine 37 to alanine (H3K37A) mutant predicted to disrupt a histone-DNA contact promotes histone degradation, hyperactivates TORC1, and causes cytotoxicity upon TORC1 inhibition. This cytotoxicity is specific to H3K37A, since chromatin instability in wild-type histone H3 (H3WT) cells reduces histone abundance and hyperactivates TORC1 without cytotoxicity upon TORC1 inhibition. Combining H3K37A with these chromatin stability mutants exacerbates histone loss, TORC1 hyperactivity, and lethality during TORC1 inhibition. Transcriptome analysis indicates H3K37A deregulates mitochondrial retrograde signaling, which is exacerbated upon TORC1 inhibition and becomes cytotoxic due in part to defective mitochondrial import of an OXPHOS subunit. Retrograde inactivation, or neutralization of reactive oxygen species (ROS) prevents this toxicity. These findings identify chromatin stability as an upstream restraint on TORC1 signaling, revealing bidirectional communication between chromatin and a major nutrient-responsive pathway. They further show H3K37 has an additional functionality that prevents mitochondrial dysregulation during metabolic stress adaptation.

Cells dynamically regulate chromatin in response to nutrient flux which promotes the transcriptional changes necessary for adaptation. The mechanistic target of rapamycin complex 1 (mTORC1) kinase integrates nutrient signaling with chromatin regulation, yet whether chromatin stability feeds back to mTORC1 activation and stress adaption remains unknown. We previously identified histone H3 at lysine 37 (H3K37) as essential for the response to mTORC1 stress such that mutation of H3K37 to alanine (H3K37A) causes cell death upon mTORC1 inhibition. Herein, we show that H3K37-dependent chromatin stability prevents proteasome-mediated histone degradation, restricts mTORC1 signaling, and safeguards mitochondrial homeostasis during mTORC1 stress. Genetic interaction analyses reveal that H3K37A combined with mutants that destabilize chromatin, including loss of the Set2 H3K36 methyltransferase, Rpd3S histone deacetylase, or multiple histone deposition pathways, causes synthetic lethality when mTORC1 is inhibited. Transcriptome analysis indicates that H3K37A misregulates the mitochondrial transcriptome during mTORC1 stress, which increases mitochondrial reactive oxygen species (ROS) and triggers lethal mitochondrial retrograde signaling. Inactivation of retrograde signaling, or ROS neutralization, rescues viability of H3K37A and chromatin stability mutants during mTORC1 stress. These findings establish chromatin stability as a key safeguard that restrains mTORC1 activity and prevents toxic mitochondrial stress during metabolic adaptation.

The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis, and endolysosomal-dependent nutrient signaling. To define how Ccr4-Not mediated ubiquitin signaling regulates these additional processes, we performed quantitative proteomics in the yeast Saccharomyces cerevisiae lacking the Not4 ubiquitin ligase, and also in cells overexpressing either wild-type or functionally inactive ligase. Herein, we provide evidence that both increased and decreased Ccr4-Not ubiquitin signaling disrupts ribosomal protein (RP) homeostasis independently of reduced RP mRNA changes or reductions in known Not4 ribosomal substrates. Surprisingly, we also find that both Not4-mediated ubiquitin signaling, and the Ccr4 subunit, actively inhibit 40S ribosomal autophagy. This 40S autophagy is independent of canonical Atg7-dependent macroautophagy, thus indicating microautophagy activation is responsible. Furthermore, the Not4 ligase genetically interacts with endolysosomal pathway effectors to control both RP expression and 40S autophagy efficiency. Overall, we demonstrate that balanced Ccr4-Not ligase activity maintains RP homeostasis, and that Ccr4-Not ubiquitin signaling interacts with the endolysosomal pathway to both regulate RP expression and inhibit 40S ribosomal autophagy.

Glioblastoma (GBM) is the most common primary brain malignancy in adults with a dismal prognosis. Despite advances in genomic analysis and surgical technique and the development of targeted therapeutics, most treatment options are ineffective and mainly palliative. Autophagy is a form of cellular self-digestion with the goal of recycling intracellular components to maintain cell metabolism. Here, we describe some recent findings that suggest GBM tumors are more sensitive to the excessive overactivation of autophagy leading to autophagy-dependent cell death. GBM cancer stem cells (GSCs) are a subset of the GBM tumor population that play critical roles in tumor formation and progression, metastasis, and relapse, and they are inherently resistant to most therapeutic strategies. Evidence suggests that GSCs are able to adapt to a tumor microenvironment of hypoxia, acidosis, and lack of nutrients. These findings have suggested that autophagy may promote and maintain the stem-like state of GSCs as well as their resistance to cancer treatment. However, autophagy is a double-edged sword and may have anti-tumor properties under certain conditions. The role of the STAT3 transcription factor in autophagy is also described. These findings provide the basis for future research aimed at targeting the autophagy-dependent pathway to overcome the inherent therapeutic resistance of GBM in general and to specifically target the highly therapy-resistant GSC population through autophagy regulation.

The cellular response to environmental exposures, such as nutrient shifts and various forms of stress, requires the integration of the signaling apparatus that senses these environmental changes with the downstream gene regulatory machinery. Delineating this molecular circuitry remains essential for understanding how organisms adapt to environmental flux, and it is critical for determining how dysregulation of these mechanisms causes disease. Ccr4-Not is a highly conserved regulatory complex that controls all aspects of the gene expression process. Recent studies in budding yeast have identified novel roles for Ccr4-Not as a key regulator of core nutrient signaling pathways that control cell growth and proliferation, including signaling through the mechanistic target of rapamycin complex 1 (TORC1) pathway. Herein, I will review the current evidence that implicate Ccr4-Not in nutrient signaling regulation, and I will discuss important unanswered questions that should help guide future efforts to delineate Ccr4-Not’s role in linking environmental signaling with the gene regulatory machinery. Ccr4-Not is highly conserved throughout eukaryotes, and increasing evidence indicates it is dysregulated in a variety of diseases. Determining how Ccr4-Not regulates these signaling pathways in model organisms such as yeast will provide a guide for defining how it controls these processes in human cells.

The Ccr4-Not complex functions as an effector of multiple signaling pathways that control gene transcription and mRNA turnover. Consequently, Ccr4-Not contributes to a diverse array of processes, which includes a significant role in cell metabolism. Yet a mechanistic understanding of how it contributes to metabolism is lacking. Herein, we provide evidence that Ccr4-Not activates nutrient signaling through the essential target of rapamycin complex 1 (TORC1) pathway. Ccr4-Not disruption reduces global TORC1 signaling, and it also upregulates expression of the cell wall integrity (CWI) pathway terminal kinase Mpk1. Although CWI signaling represses TORC1 signaling, we find that Ccr4-Not loss inhibits TORC1 independently of CWI activation. Instead, we demonstrate that Ccr4-Not promotes the function of the vacuole V-ATPase, which interacts with the Gtr1 GTPase-containing EGO complex to stimulate TORC1 in response to nutrient sufficiency. Bypassing the V-ATPase requirement in TORC1 activation using a constitutively active Gtr1 mutant fully restores TORC1 signaling in Ccr4-Not deficient cells. Transcriptome analysis and functional studies revealed that loss of the Ccr4 subunit activates the TORC1 repressed retrograde signaling pathway to upregulate mitochondrial activity. Blocking this mitochondrial upregulation in Ccr4-Not deficient cells further represses TORC1 signaling, and it causes synergistic deficiencies in mitochondrial-dependent metabolism. These data support a model whereby Ccr4-Not loss impairs V-ATPase dependent TORC1 activation that forces cells to enhance mitochondrial metabolism to sustain a minimal level of TORC1 signaling necessary for cell growth and proliferation. Therefore, Ccr4-Not plays an integral role in nutrient signaling and cell metabolism by promoting V-ATPase dependent TORC1 activation. Eukaryotic cells dynamically control nutrient signaling pathways to regulate metabolism, anabolic growth, and cell proliferation through molecular effectors that remain poorly understood. The Ccr4-Not complex is a multiprotein regulatory factor conserved from yeast to humans that integrates both mRNA degradation and ubiquitin ligase activities. While budding yeast Ccr4-Not represses mitochondrial metabolism to allow normal glycolytic metabolism, how it does so is not clear. Herein, we demonstrate that Ccr4-Not activates nutrient signaling through the conserved target of rapamycin complex 1 (TORC1) pathway by promoting the function of the vacuolar V-ATPase proton pump that acidifies the vacuole and activates TORC1. Ccr4-Not deficient cells exhibit decreased V-ATPase stability, leading to defects in both vacuole acidification and TORC1 signaling. This TORC1 defect causes an adaptive upregulation of mitochondrial metabolism to maintain a minimal level of TORC1 activity. This study defines a previously unknown role for Ccr4-Not as a key activator of a conserved eukaryotic nutrient signaling pathway highly relevant to human health and disease.

The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.

The Ccr4-Not complex controls RNA polymerase II (Pol II) dependent gene expression and proteasome function. The Not4 ubiquitin ligase is a Ccr4-Not subunit that has both a RING domain and a conserved RNA recognition motif and C3H1 domain (referred to as the RRM-C domain) with unknown function. We demonstrate that while individual Not4 RING or RRM-C mutants fail to replicate the proteasomal defects found in Not4 deficient cells, mutation of both exhibits a Not4 loss of function phenotype. Transcriptome analysis revealed that the Not4 RRM-C affects a specific subset of Pol II-regulated genes, including those involved in transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. The Not4 RING, RRM-C, or RING/RRM-C mutations cause a generalized increase in Pol II binding at a subset of these genes, yet their impact on gene expression does not always correlate with Pol II recruitment which suggests Not4 regulates their expression through additional mechanisms. Intriguingly, we find that while the Not4 RRM-C is dispensable for Ccr4-Not association with RNA Pol II, the Not4 RING domain is required for these interactions. Collectively, these data elucidate previously unknown roles for the conserved Not4 RRM-C and RING domains in regulating Ccr4-Not dependent functionsin vivo.