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T Keith Blackwell

Publications and source records attributed to T Keith Blackwell.

At least 19 recordsLinked to original sources
PubMed2006

A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span.

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. Here, we demonstrate that the protein kinase MST1 mediates oxidative-stress-induced cell death in primary mammalian neurons by directly activating the FOXO transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the forkhead domain that disrupts their interaction with 14-3-3 proteins, promotes FOXO nuclear translocation, and thereby induces cell death in neurons. We also extend the MST-FOXO signaling link to nematodes. Knockdown of the C. elegans MST1 ortholog CST-1 shortens life span and accelerates tissue aging, while overexpression of cst-1 promotes life span and delays aging. The cst-1-induced life-span extension occurs in a daf-16-dependent manner. The identification of the FOXO transcription factors as major and evolutionarily conserved targets of MST1 suggests that MST kinases play important roles in diverse biological processes including cellular responses to oxidative stress and longevity.

14-3-3 Proteins↗
PubMed2005

Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3.

Oxidative stress plays a central role in many human diseases and in aging. In Caenorhabditis elegans the SKN-1 protein induces phase II detoxification gene transcription, a conserved oxidative stress response, and is required for oxidative stress resistance and longevity. Oxidative stress induces SKN-1 to accumulate in intestinal nuclei, depending on p38 mitogen-activated protein kinase signaling. Here we show that, in the absence of stress, phosphorylation by glycogen synthase kinase-3 (GSK-3) prevents SKN-1 from accumulating in nuclei and functioning constitutively in the intestine. GSK-3 sites are conserved in mammalian SKN-1 orthologs, indicating that this level of regulation may be conserved. If inhibition by GSK-3 is blocked, background levels of p38 signaling are still required for SKN-1 function. WT and constitutively nuclear SKN-1 comparably rescue the skn-1 oxidative stress sensitivity, suggesting that an inducible phase II response may provide optimal stress protection. We conclude that (i) GSK-3 inhibits SKN-1 activity in the intestine, (ii) the phase II response integrates multiple regulatory signals, and (iii), by inhibiting this response, GSK-3 may influence redox conditions.

Amino Acid Sequence↗
PubMed2005

A conserved RNA-protein complex component involved in physiological germline apoptosis regulation in C. elegans.

Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific ;physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. We conclude that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. We propose that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis.

Animals↗
PubMed2005

The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response.

The evolutionarily conserved p38 mitogen-activated protein kinase (MAPK) cascade is an integral part of the response to a variety of environmental stresses. Here we show that the Caenorhabditis elegans PMK-1 p38 MAPK pathway regulates the oxidative stress response via the CNC transcription factor SKN-1. In response to oxidative stress, PMK-1 phosphorylates SKN-1, leading to its accumulation in intestine nuclei, where SKN-1 activates transcription of gcs-1, a phase II detoxification enzyme gene. These results delineate the C. elegans p38 MAPK signaling pathway leading to the nucleus that responds to oxidative stress.

Active Transport, Cell Nucleus↗
PubMed2005

Requirement for P granules and meiosis for accumulation of the germline RNA helicase CGH-1.

In Caenorhabditis elegans, lack of the conserved germline RNA helicase CGH-1 causes infertility and excessive levels of physiological germline apoptosis, a process that normally claims about half of all developing oocytes. In yeast the CGH-1 ortholog is a key component of degradative "processing (P) bodies," which may share some properties with germline protein-RNA complexes such as P granules. During oogenesis CGH-1 associates with P granules, but also accumulates to high levels in additional cytoplasmic particles. Here we show that appropriate levels and localization of CGH-1 depends on some P granule components and on mechanisms that establish meiotic development. At the same time, germ cell death is not increased by various abnormalities in P granules or meiosis. We conclude that in developing oocytes CGH-1 particles accumulate specifically in response to meiotic development and have distinct functions from P granules, and may be dynamic protein-mRNA structures.

Animals↗
PubMed2004

The Caenorhabditis elegans ortholog of TRAP240, CeTRAP240/let-19, selectively modulates gene expression and is essential for embryogenesis.

Mediator complexes are large multiprotein assemblies that function in the regulation of eukaryotic gene transcription. In yeast, certain mediator subunits appear to comprise a subcomplex that acts in the regulation of a specific subset of genes. We investigated in a metazoan, Caenorhabditis elegans, the roles and interactions of two of those subunits, CeTRAP240/let-19 and CeTRAP230/dpy-22. We found that CeTRAP240/let-19 contains four domains that are conserved in the human TRAP240 protein and that one of those domains displays intrinsic transcriptional repression activity. Using RNA interference, we found that reduced expression of CeTRAP240/let-19 displayed a high penetrance of embryonic lethality in F1 progeny; animals that escaped embryonic arrest showed mutant phenotypes such as burst vulva and molting defects. CeTRAP240/let-19 appeared to affect specific genes, as CeTRAP240/let-19(RNAi) led to selectively reduced expression of a subset of reporter genes examined. Genetic experiments supported the view that CeTRAP240/let-19 and CeTRAP230/dpy-22, like their Drosophila and yeast counterparts, can operate on common pathways. Thus, a male tail phenotype caused by the pal-1(e2091) mutation was suppressed not only by CeTRAP230/dpy-22 mutants, as reported previously, but also by reduced expression of CeTRAP240/let-19. Additionally, CeTRAP240/let-19(RNAi) in a CeTRAP230/dpy-22 mutant background produced a strong synthetic lethal phenotype. Overall, our results establish specific roles of CeTRAP240/let-19 in C. elegans embryonic development and a functional interaction between CeTRAP240/let-19 and CeTRAP230/dpy-22. Interestingly, whereas this interaction has been conserved from yeast to mammals, the subcomplex modulates metazoan-specific genetic pathways, likely in addition to those also controlled in yeast.

Amino Acid Sequence↗
PubMed2004

Germ cells: finding programs of mass repression.

Transcription is globally silenced in the germline of animals. Recent studies have shown that, in Caenorhabditis elegans, this silencing is initially mediated through direct repression, but in Drosophila, the factors involved include pgc, a non-coding cytoplasmic RNA. Why are these mechanisms so diverse and complex?

Animals↗
PubMed2004

MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay.

Stress granules (SGs) are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. SG-associated proteins such as TIA-1, TIAR and HuR bind to AU-rich element (ARE)-containing mRNAs and control their translation and stability. Here we show that tristetraprolin (TTP), an ARE-binding protein that destabilizes ARE-mRNAs, is recruited to SGs that are assembled in response to FCCP-induced energy deprivation, but not arsenite-induced oxidative stress. Exclusion of TTP from arsenite-induced SGs is a consequence of MAPKAP kinase-2 (MK2)-induced phosphorylation at serines 52 and 178, which promotes the assembly of TTP:14-3-3 complexes. 14-3-3 binding excludes TTP from SGs and inhibits TTP-dependent degradation of ARE-containing transcripts. In activated RAW 264.7 macrophages, endogenous TTP:14-3-3 complexes bind to ARE-RNA. Our data reveal the mechanism by which the p38-MAPK/MK2 kinase cascade inhibits TTP-mediated degradation of ARE-containing transcripts and thereby contributes to lipopolysaccharide-induced TNFalpha expression.

14-3-3 Proteins↗
PubMed2004

An extensive requirement for transcription factor IID-specific TAF-1 in Caenorhabditis elegans embryonic transcription.

The general transcription factor TFIID sets the mRNA start site and consists of TATA-binding protein and associated factors (TAF(II)s), some of which are also present in SPT-ADA-GCN5 (SAGA)-related complexes. In yeast, results of multiple studies indicate that TFIID-specific TAF(II)s are not required for the transcription of most genes, implying that intact TFIID may have a surprisingly specialized role in transcription. Relatively little is known about how TAF(II)s contribute to metazoan transcription in vivo, especially at developmental and tissue-specific genes. Previously, we investigated functions of four shared TFIID/SAGA TAF(II)s in Caenorhabditis elegans. Whereas TAF-4 was required for essentially all embryonic transcription, TAF-5, TAF-9, and TAF-10 were dispensable at multiple developmental and other metazoan-specific promoters. Here we show evidence that in C. elegans embryos transcription of most genes requires TFIID-specific TAF-1. TAF-1 is not as universally required as TAF-4, but it is essential for a greater proportion of transcription than TAF-5, -9, or -10 and is important for transcription of many developmental and other metazoan-specific genes. TAF-2, which binds core promoters with TAF-1, appears to be required for a similarly substantial proportion of transcription. C. elegans TAF-1 overlaps functionally with the coactivator p300/CBP (CBP-1), and at some genes it is required along with the TBP-like protein TLF(TRF2). We conclude that during C. elegans embryogenesis TAF-1 and TFIID have broad roles in transcription and development and that TFIID and TLF may act together at certain promoters. Our findings imply that in metazoans TFIID may be of widespread importance for transcription and for expression of tissue-specific genes.

Animals↗
PubMed2003

SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.

During the earliest stages of Caenorhabditis elegans embryogenesis, the transcription factor SKN-1 initiates development of the digestive system and other mesendodermal tissues. Postembryonic SKN-1 functions have not been elucidated. SKN-1 binds to DNA through a unique mechanism, but is distantly related to basic leucine-zipper proteins that orchestrate the major oxidative stress response in vertebrates and yeast. Here we show that despite its distinct mode of target gene recognition, SKN-1 functions similarly to resist oxidative stress in C. elegans. During postembryonic stages, SKN-1 regulates a key Phase II detoxification gene through constitutive and stress-inducible mechanisms in the ASI chemosensory neurons and intestine, respectively. SKN-1 is present in ASI nuclei under normal conditions, and accumulates in intestinal nuclei in response to oxidative stress. skn-1 mutants are sensitive to oxidative stress and have shortened lifespans. SKN-1 represents a connection between developmental specification of the digestive system and one of its most basic functions, resistance to oxidative and xenobiotic stress. This oxidative stress response thus appears to be both widely conserved and ancient, suggesting that the mesendodermal specification role of SKN-1 was predated by its function in these detoxification mechanisms.

Amino Acid Motifs↗
PubMed2003

A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb.

The positive transcription elongation factor b (P-TEFb) contains cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9). For activating the expression of eukaryotic genes, the histidine-rich sequence in CycT1 binds the heptapeptide repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), whereupon Cdk9 phosphorylates the CTD. We found that alanine-substituted heptapeptide repeats that cannot be phosphorylated also bind CycT1. When placed near transcription units, these CTD analogs block effects of P-TEFb. Remarkably, the transcriptional repressor PIE-1 from Caenorhabditis elegans behaves analogously. It binds CycT1 via an alanine-containing heptapeptide repeat and inhibits transcriptional elongation. Thus, our findings reveal a new mechanism by which repressors inhibit eukaryotic transcription.

Alanine↗
PubMed2003

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization.

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.

Amino Acid Sequence↗
PubMed2002

A broad but restricted requirement for TAF-5 (human TAFII100) for embryonic transcription in Caenorhabditis elegans.

As conserved components of the transcription factor (TF) IID- and TFTC/SAGA-related complexes, TATA-binding protein-associated factors (TAF(II)s) are important for eukaryotic mRNA transcription. In yeast, genetic analyses suggest that, although some individual TAF(II)s are required for transcription of most genes, others have highly specialized functions. Much less is known about the functions of TAF(II)s in metazoans, which have more complex genomes that include many tissue-specific genes. TAF-5 (human (h) TAF(II)100) is of particular interest because it is predicted to have an important structural role. Here we describe the first genetics-based analysis of TAF-5 in a metazoan. By performing RNA interference in Caenorhabditis elegans embryos, which can survive for several cell generations without transcription, we found that taf-5 is important for a significant fraction of transcription. However, TAF-5 is apparently not essential for the expression of multiple developmental and other metazoan-specific genes. This phenotype remarkably resembles the previously described effects of similarly depleting two C. elegans histone fold TAF(II)s, TAF-9 (hTAF(II)31/32) and TAF-10 (hTAF(II)30), but is distinct from the widespread transcription block caused by TAF-4 (hTAF(II)130) depletion. Our findings suggest that TAF-5, TAF-9, and TAF-10 are part of a functional module of TFIID- and TFTC/SAGA-related complexes that can be bypassed in many metazoan-specific genes.

Amino Acid Sequence↗
PubMed2002

CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo.

The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Here we used RNA interference to investigate P-TEFb functions in vivo, in the Caenorhabditis elegans embryo. We found that P-TEFb is broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or insure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways.

Animals↗
PubMed2002

Broad requirement for the mediator subunit RGR-1 for transcription in the Caenorhabditis elegans embryo.

The Mediator-related transcription co-factors integrate positive and negative inputs and recruit and activate the RNA polymerase II complex. To understand the role of Mediator during transcription, it is important to identify Mediator subunits that are essential for its functions. In the yeast Mediator, the conserved component Rgr1 is associated with multiple subunits that are required for specific activation or repression events. Yeast rgr1 is essential for viability, for certain repression mechanisms, and for activation of heat shock genes, but it is not known whether rgr1 is generally important for transcription. Here we have performed the first analysis of rgr-1 function in a metazoan. We found that in the developing Caenorhabditis elegans embryo rgr-1 is broadly required for transcription and for phosphorylation of both Ser-2 and Ser-5 of the RNA polymerase II C-terminal domain repeat. We conclude that RGR-1 fulfills a critical Mediator function that is broadly essential for metazoan mRNA transcription and that RGR-1 may be required at an early recruitment or initiation step.

Amino Acid Sequence↗