skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Thursday, May 23 until 2:00 AM ET on Friday, May 24 due to maintenance. We apologize for the inconvenience.


Title: CCR4‐NOT subunit CCF ‐1/ CNOT7 promotes transcriptional activation to multiple stress responses in Caenorhabditis elegans
Abstract

CCR4‐NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4‐NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome‐wide RNAi screen, we identified a subunit of the CCR4‐NOT complex,ccf‐1, as a requirement for theC. eleganstranscriptional response to cadmium and acrylamide stress. Using whole‐transcriptome RNA sequencing, we show that the knockdown ofccf‐1attenuates the activation of a broad range of stress‐protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown ofccf‐1decreasesC. elegansstress resistance and normal lifespan. A yeast 2‐hybrid screen using a CCF‐1 bait identified the homeobox transcription factor PAL‐1 as a physical interactor. Knockdown ofpal‐1inhibits the activation ofccf‐1 dependent stress genes and reducesC. elegansstress resistance. Gene expression analysis reveals that knockdown ofccf‐1andpal‐1attenuates the activation ofelt‐2andelt‐3under stress that encode master transcriptional co‐regulators of stress response in theC. elegans, and that overexpression of ELT‐2 can suppressccf‐1's requirement for gene transcription in a stress‐dependent manner. Our findings reveal a new role for CCR4‐NOT in the environmental stress response and define its role in stress resistance and longevity inC. elegans.

 
more » « less
NSF-PAR ID:
10420259
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Aging Cell
Volume:
22
Issue:
4
ISSN:
1474-9718
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender] 
    more » « less
  2. Abstract Background

    TheBIN1locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease.BIN1undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth.

    Methods

    Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells.Bin1expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches.

    Results

    Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencingBin1expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specificBin1conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss ofBin1impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene,Ifitm3.

    Conclusions

    Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship betweenBin1andIfitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulateIfitm3upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.

    Graphical Abstract 
    more » « less
  3. Summary

    Aging is associated with a large number of both phenotypic and molecular changes, but for most of these, it is not known whether these changes are detrimental, neutral, or protective. We have identified a conservedCaenorhabditis elegansGATA transcription factor/MTA‐1 homologegr‐1(lin‐40) that extends lifespan and promotes resistance to heat and UV stress when overexpressed. Expression ofegr‐1increases with age, suggesting that it may promote survival during normal aging. This increase in expression is dependent on the presence of the germline, raising the possibility thategr‐1expression is regulated by signals from the germline. In addition, loss ofegr‐1suppresses the long lifespan of insulin receptordaf‐2mutants. The DAF‐16 FOXO transcription factor is required for the increased stress resistance ofegr‐1overexpression mutants, andegr‐1is necessary for the proper regulation ofsod‐3(a reporter for DAF‐16 activity). These results indicate thategr‐1acts within the insulin signaling pathway.egr‐1can also activate the expression of its paralogegl‐27,another factor known to extend lifespan and increase stress resistance, suggesting that the two genes act in a common program to promote survival. These results identifyegr‐1as part of a longevity‐promoting circuit that changes with age in a manner that is beneficial for the lifespan of the organism.

     
    more » « less
  4. Abstract

    Gene regulation in changing environments is critical for maintaining homeostasis. Some animals undergo a stress-resistant diapause stage to withstand harsh environmental conditions encountered during development. MicroRNAs are one mechanism for regulating gene expression during and after diapause. MicroRNAs downregulate target genes posttranscriptionally through the activity of the microRNA-induced silencing complex. Argonaute is the core microRNA-induced silencing complex protein that binds to both the microRNA and to other microRNA-induced silencing complex proteins. The 2 major microRNA Argonautes in the Caenorhabditis elegans soma are ALG-1 and ALG-2, which function partially redundantly. Loss of alg-1 [alg-1(0)] causes penetrant developmental phenotypes including vulval defects and the reiteration of larval cell programs in hypodermal cells. However, these phenotypes are essentially absent if alg-1(0) animals undergo a diapause stage called dauer. Levels of the relevant microRNAs are not higher during or after dauer, suggesting that activity of the microRNA-induced silencing complex may be enhanced in this context. To identify genes that are required for alg-1(0) mutants to develop without vulval defects after dauer, we performed an RNAi screen of genes encoding conserved kinases. We focused on kinases because of their known role in modulating microRNA-induced silencing complex activity. We found RNAi knockdown of 4 kinase-encoding genes, air-2, bub-1, chk-1, and nekl-3, caused vulval defects and reiterative phenotypes in alg-1(0) mutants after dauer, and that these defects were more penetrant in an alg-1(0) background than in wild type. Our results implicate these kinases as potential regulators of microRNA-induced silencing complex activity during postdauer development in C. elegans.

     
    more » « less
  5. Summary

    Emerging evidence indicates a close connection between cell‐cycle progression and the plant immune responses. In Arabidopsis,MODIFIER OFsnc1‐1(MOS1) modulates a number of processes including endoreduplication and plant disease resistance, but the molecular mechanism underlying this modulation was not fully understood. Here, we provide biochemical and genetic evidence thatTEOSINTE BRANCHED1,CYCLOIDEA,PCF1 (TCP) transcription factorsTCP15 and its homologues are mediators ofMOS1 function in the immune response and are likely to be also involved in cell‐cycle control.MOS1 andTCPproteins have a direct physical interaction. They both bind to the promoter of the immune receptor geneSUPRESSOR OFnpr1‐1,CONSTITUTIVE1(SNC1) and modulate its expression and consequently immune responses.MOS1 andTCP15 both affect the expression of cell‐cycle genesD‐typeCYCLIN3;1(CYCD3;1), which may mediate theMOS1 function in cell‐cycle modulation. In addition,CYCD3;1overexpression upregulates immune responses, andSNC1expression. This study investigated and revealed a role forMOS1 in transcriptional regulation throughTCP15 and its homologues. This finding suggests the coordination of cell‐cycle progression and plant immune responses at multiple levels.

     
    more » « less