T. Keith Blackwell et al. (2006) WormBook "Transcription mechanisms."

Amy K. Walker, T. Keith Blackwell

[WormBook, 2006]

Appropriate regulation of mRNA transcription is central to the differentiation and functions of eukaryotic cells, and to the development of complex organisms. mRNAs are synthesized by the coordinated action of a set of general transcription and mRNA modification factors. These factors and the fundamental mechanisms involved in transcription are conserved among eukaryotes, including C. elegans. Recent studies in various systems have revealed that this apparatus is not controlled through a simple on/off switch at the promoter, and that the factors and mechanisms involved in transcription are instead subject to regulation at a surprising number of different levels. In this chapter we will discuss examples in which regulation involving the general mRNA transcription apparatus or other transcription co-factors plays a central role in C. elegans development, and in which C. elegans studies have provided new insights into eukaryotic transcription mechanisms. Together, these studies have shown that regulatory mechanisms that involve the general Pol II machinery are a central participant in many aspects of C. elegans biology.

Isik, Meltem et al. (2015) International Worm Meeting "Investigation of TORC1-inhibition dependent SKN-1/Nrf activity and its effect on longevity ."

Isik, Meltem, Blackwell, Keith T

[International Worm Meeting, 2015]

The target of rapamycin (TOR) signaling is one of the pathways that regulate cell growth and metabolism in response to food availability and it has been broadly implicated in aging. Rapamycin, a TOR inhibitor, is the only drug studied so far that has unambiguously been shown to slow mammalian aging. However, in addition to its beneficial effects, it comes with harmful secondary effects, such as diabetic symptoms and suppression of immune system. It is therefore critical to identify specific mechanisms downstream of rapamycin that affect aging. The findings of our lab suggest that SKN-1/Nrf transcription factor is required for lifespan extensions associated with genetic inhibition of either TORC1 or TORC2, or rapamycin treatment indicating that it plays a central role in the influence of TOR pathways on aging. Interference with TOR signaling results in increased SKN-1/Nrf induced transcription, which seems to be critical for its effects on aging and stress resistance. Therefore, novel findings about the TORC1-SKN-1/Nrf-longevity axis might be useful for development of agents that promote particular SKN-1/Nrf activities, and would specifically reinforce beneficial effects of TOR inhibition by avoiding unwanted effects related to immunity, insulin sensitivity or inhibition of critical TOR functions. Here, we used high-throughput RNAi screens to determine the genes that have an effect on SKN-1 activity and also interacts with TOR signaling pathway. The tested 1451 clones consisted of genes that are available in TOR phosphoproteome data from six previous studies done in mammalian and yeast cells and the related genes in associated pathways. Each of these clones were tested for their effect on expression of a SKN-1/Nrf target gene reporter, Pgst-4::gfp, in WT, raga-1(ok386) (a TORC1 inhibition background) and skn-1(lax188) gain of function worms. This analysis has revealed already known as well as previously unidentified activators and repressors of SKN-1/Nrf activity, which also phenotypically interacted with TOR signaling pathway. Highly represented pathways included lin-35/Rb, chromatin factors, ubiquitin-proteosome, RNA processing/spliceosome, and endocytosis pathways. Therefore, these identified pathways can be responsible for TOR inhibition dependent SKN-1/Nrf activity and related genes will be analyzed further for their effect on lifespan and stress resistance of C. elegans. The findings of this research study may lead to development of agents that specifically reinforce beneficial effects of TOR inhibition by avoiding unwanted effects.

Wu, Ziyun et al. (2017) International Worm Meeting "Longevity from dietary restriction and reduced insulin/IGF-1 signaling requires modulation of p38/ATF-7 innate immunity."

Blackwell, Keith, Steinbaugh, Michael, Wu, Ziyun, Isik, Meltem, Moroz, Natalie

[International Worm Meeting, 2017]

Dietary restriction (DR) and reduced insulin/IGF-1 signaling (rIIS) are the most robust environmental and genetic interventions, respectively, that to extend healthy lifespan in diverse organisms. In mammals, through unknown mechanisms DR reduces chronic inflammation, a cause of aging-related disease. Here we show that in C. elegans, DR and rIIS promote longevity by modulating innate immunity. For DR and rIIS lifespan extension, the conserved innate immunity pathway TIR-1(SARM)-NSY-1(ASK1)- SEK-1(MKK3)-PMK-1(p38)-ATF-7(ATF2) must be intact. However, in contrast to other protective mechanisms, p38/ATF-7 immune activity is reduced to a basal level by DR and rIIS, which mobilize other pathogen resistance programs. Elevated p38/ATF-7 immunity accelerates aging, but can be reversed by DR or rIIS to allow lifespan extension. We conclude that innate immunity modulation is critical for longevity assurance, and that regulation of immunity by anabolic signals may have contributed to immune system evolution.

Moroz, Natalie et al. (2015) International Worm Meeting "Dietary restriction involves NAD+-dependent mechanisms and a shift towards oxidative metabolism."

Hart, Anne, Sinclair, David, Blackwell, Keith, Anderson, Edward, Carmona, Juan, Moroz, Natalie

[International Worm Meeting, 2015]

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD+-dependent deacylases known as sirtuins, and increases in NAD+ that derive from higher levels of respiration. Here we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens (DAF-16 (FOXO), SKN-1 (Nrf1/2/3), PHA-4 (FOXA), AAK-2 (AMPK)). Complete DR lifespan extension required the sirtuin SIR-2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC-1, a key NAD+ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc-1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD+-dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD+ salvage. Under DR conditions, NAD+-dependent processes may be supported by a DR-induced shift towards oxidative metabolism rather than an increase in total respiration. JC and EA contributed equally. AH, DS, and KB are co-corresponding authors.

Campos, Juliane et al. (2021) International Worm Meeting "Mild impairment of mitochondrial function increases longevity and pathogen resistance through ATFS-1-driven activation of p38-regulated innate immunity"

Blackwell, Keith, Mistry, Meeta, Ferreira, Julio, Campos, Juliane, Wu, Ziyun, Rudich, Paige, Van Raamsdonk, Jeremy, Soo, Sonja

[International Worm Meeting, 2021]

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain resulted in upregulation of genes involved in innate immunity, which we found to be dependent on not only the canonical p38-mediated innate immune signaling pathway but also on the mitochondrial unfolded protein response. Both of these pathways are absolutely required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitochondrial unfolded protein response can act on the same innate immunity genes to promote resistance to bacterial pathogens, and that input from the mitochondria can extend longevity by signaling through these two pathways. Combined, this indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

Ji Yun Hwang et al. (2007) International Worm Meeting "The intestinal expression of gst-7 is regulated by insulin/IGF-1 signaling pathway in skn-1 dependent manner but not daf-16."

T. Keith Blackwell, Jae Hyung An, Ji Yun Hwang, Yu Sam Kim, Young-Ki Paik, Jeeyong Lee

[International Worm Meeting, 2007]

Oxidative stress has been implicated in human disease and it is also important in aging. However, cells have multiple protective mechanisms against damage resulting from oxidative stress. In C. elegans, SKN-1 is required for oxidative stress resistance and for longevity (An and Blackwell (2003) Genes Dev., 17, 1882). In response to multiple stress signals, SKN-1 localizes to intestinal nuclei and induces expression of gcs-1, which catalyses glutathione biosynthesis. Both SKN-1 nuclear localization and gcs-1 induction are inhibited constitutively by GSK-3, and require p38 signaling (An et al (2005) PNAS., 102, 16275; Inoue et al (2005) Genes Dev., 19, 2278). It is well established in C. elegans that the increase in stress resistance and longevity caused by genetic inhibition of DAF-2 signaling is dependent on DAF-16. However, recent data from the Blackwell lab reveals that SKN-1 is also negatively regulated by the DAF-2 pathway, in parallel to DAF-16. As an antioxidant response gene, Glutathione S Transferase-7 (gst-7) is hypothesized to be a downstream target gene of SKN-1, and to function in oxidative stress response pathway. To test this hypothesis and identify genetic interactions, we examined how RNAi phenotypes of genes that are predicted to be upstream of gst-7 vary in gst-7 transgenic worms. We have observed that under both normal and oxidative stress conditions, the intestinal expression of GST-7 is significantly suppressed in worms that are exposed to skn-1 RNAi, but daf-16 RNAi shows no effect. Additionally, daf-2 RNAi elevates the intestinal expression of GST-7, dependent upon skn-1. Therefore, these results suggest that GST-7 is under the control of the DAF-2 pathway dependent upon skn-1 but not daf-16, supporting the recent model that DAF-2 pathway also regulates SKN-1 at least in part directly, in parallel to DAF-16.

Meng, Jin et al. (2021) International Worm Meeting "Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans"

Blackwell, Keith, Yang, Jing, Wu, Ziyun, Carroll , Kate, Meng, Jin, Fu, Ling, Liu, Keke, B. Ferreira, Renan, Tian, Caiping, Jung, Youngeun

[International Worm Meeting, 2021]

Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

Jin, CongYu et al. (2011) International Worm Meeting "Searching for transcriptional regulators of C. elegans proteasome subunits."

Kohara, Yuji, Jin, CongYu, Blackwell, Keith, Li, Xuan, Mori, Akihiro, Yang, YueHong, Dethlefsen, Johan, Holmberg, Carina I., Henriksson, Johan, Burglin, Thomas

[International Worm Meeting, 2011]

The 26S proteasome is a highly conserved multi-subunit protease that controls protein stability in the cell. It degrades damaged and misfolded proteins, as well as redundant key regulatory proteins, and is therefore involved in multiple cellular processes such as cell cycle control, signal transduction, stress response, and apoptosis. So far, the transcriptional regulation of the proteasome complex has not been well characterized, especially in multi-cellular organisms. By using transcriptional GFP reporters for several proteasome subunit genes and RNAi assays, we have established the existence of a compensatory "bounce-back" response in C. elegans, namely the up-regulation of various proteasome subunit genes in response to proteasome inhibition. We found that this feed-back response is mostly dependent on the transcription factor SKN-1, the C. elegans ortholog of mammalian Nrf1/2/3 proteins that have been recently found to mediate a similar response in mammalian cell cultures. However, RNAi targeting skn-1 alone did not have a major effect on expression levels of various proteasome subunits, suggesting that SKN-1 is mainly serving as a transcriptional regulator of proteasome subunit genes under stress conditions. In order to identify transcription factors that contribute to proteasome subunit expression under normal conditions, we used in silico approaches to search for binding sites of known transcription factors and novel cis-regulatory modules in promoter regions of various proteasome subunits. We have identified an 11-bp module that exists in promoter regions of multiple proteasome subunits. Deletion and/or substitution of the candidate module in several proteasome subunit transcriptional GFP reporters caused alterations in fluorescence intensity and distribution. Our data indicate that the predicted module serves as a cis-regulatory element of various proteasome subunit genes. We are currently looking for the transcription factor that binds to this element. Taken together, out data suggest that multiple transcription factors contribute to transcriptional regulation of proteasome gene expression, both under normal and stress conditions.

Williams CL et al. (2008) Mol Biol Cell "Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis."

Yoder BK, Winkelbauer ME, Schafer JC, Michaud EJ, Williams CL

[Mol Biol Cell, 2008]

Monitoring Editor: Keith Mostov Meckel-Gruber Syndrome (MKS), Nephronophthisis (NPHP), and Joubert Syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

Chen CC et al. (2006) Mol Biol Cell "RAB-10 is required for endocytic recycling in the Caenorhabditis elegans intestine."

Schweinsberg PJ, Chen CC, Vashist S, Mareiniss DP, Grant BD, Lambie EJ

[Mol Biol Cell, 2006]

Monitoring Editor: Keith Mostov The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid from endosomes to the plasma membrane. We have previously shown that the EH-domain protein RME-1 is a critical regulator of endocytic recycling in worms and mammals. Here we identify the RAB-10 protein as a key regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the C. elegans intestine. rab-10 null mutant intestinal cells accumulate abnormally abundant RAB-5 positive early endosomes, some of which are enlarged by more than 10-fold. Conversely most RME-1 positive recycling endosomes are lost in rab-10 mutants. The abnormal early endosomes in rab-10 mutants accumulate basolaterally recycling transmembrane cargo molecules, and basolaterally recycling fluid, consistent with a block in basolateral transport. These results indicate a role for RAB-10 in basolateral recycling upstream of RME-1. We found that a functional GFP-RAB-10 reporter protein is localized to endosomes and Golgi in wild-type intestinal cells consistent with a direct role for RAB-10 in this transport pathway.