Purification and Characterization of the Human Elongator Complex

Hawkes, Nicola A.; Otero, Gabriel; Winkler, G. Sebastiaan; Marshall, Nicholas F.; Dahmus, Michael E.; Krappmann, Daniel; Scheidereit, Claus; Thomas, Claire Le; Schiavo, Giampietro; Erdjument‐Bromage, Hediye; Tempst, Paul; Svejstrup, Jesper Q.

doi:10.1074/jbc.m110445200

Cited by 234 publications

(236 citation statements)

References 25 publications

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“…The elongator complex comprises six proteins, ELP1-6, and is responsible both for histone acetylation to facilitate transcription and for the modification of the wobble position U-34 of eukaryotic tRNAs by carbamoylmethylation or methoxy-carbonylmethylation at C-5 of uridine (35,36). ELP3 contains both radical SAM and histone acetyltransferase domains.…”

Section: Discussionmentioning

confidence: 99%

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Makins

Ghosh²,

Román‐Meléndez³

et al. 2016

Journal of Biological Chemistry

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Edited by Charles SamuelViperin is an endoplasmic reticulum-associated antiviral responsive protein that is highly up-regulated in eukaryotic cells upon viral infection through both interferon-dependent and independent pathways. Viperin is predicted to be a radical S-adenosyl-L-methionine (SAM) enzyme, but it is unknown whether viperin actually exploits radical SAM chemistry to exert its antiviral activity. We have investigated the interaction of viperin with its most firmly established cellular target, farnesyl pyrophosphate synthase (FPPS). Numerous enveloped viruses utilize cholesterol-rich lipid rafts to bud from the host cell membrane, and it is thought that by inhibiting FPPS activity (and therefore cholesterol synthesis), viperin retards viral budding from infected cells. We demonstrate that, consistent with this hypothesis, overexpression of viperin in human embryonic kidney cells reduces the intracellular rate of accumulation of FPPS but does not inhibit or inactivate FPPS. The endoplasmic reticulum-localizing, N-terminal amphipathic helix of viperin is specifically required for viperin to reduce cellular FPPS levels. However, although viperin reductively cleaves SAM to form 5-deoxyadenosine in a slow, uncoupled reaction characteristic of radical SAM enzymes, this cleavage reaction is independent of FPPS. Furthermore, mutation of key cysteinyl residues ligating the catalytic [Fe 4 S 4 ] cluster in the radical SAM domain, surprisingly, does not abolish the inhibitory activity of viperin against FPPS; indeed, some mutations potentiate viperin activity. These observations imply that viperin does not act as a radical SAM enzyme in regulating FPPS. Radical S-adenosyl-L-methionine-dependent (SAM)3 enzymes constitute a superfamily of enzymes that use SAM to generate free radicals (1-4). The superfamily is typified by a common CXXXCXXC motif, the cysteinyl residues of which coordinate a [Fe 4 S 4 ] cluster that is essential for radical generation. These enzymes catalyze a remarkably wide range of reactions involving an equally diverse set of substrates (2). For example, radical SAM-dependent enzymes participate in the biosynthesis of herbicides, antibiotics, vitamins, co-factors such as biotin and thiamin, and various other natural products (2, 5-8). They also function in the modification of ribosomal and transfer RNAs (9, 10) and DNA repair (11) and in the post-translational modification of peptides and proteins (12)(13)(14). Sequence analyses indicate that there are potentially thousands of members of the radical SAM superfamily, but to date, relatively few of these enzymes have been isolated and characterized (15, 16). Radical SAM enzymes were until recently thought to be confined to the microbial realm but intriguingly have now been identified in higher aerobic organisms, including plants and animals (3).Central to the mechanism of all radical SAM enzymes is the generation of a highly reactive 5Ј-deoxyadenosyl radical (Ado ⅐ ) (1,4,17). This is accomplished through one-electron reduction of SAM by the [Fe 4...

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Section: Discussionmentioning

confidence: 99%

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Makins

Ghosh²,

Román‐Meléndez³

et al. 2016

Journal of Biological Chemistry

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“…Leaf mutants with narrow and elongated lamina shape allowed the identification of the plant homologs of the yeast Elongator complex components (8). In yeast and humans, Elongator consists of the core subcomplex composed of ELP1, ELP2, and ELP3 and the accessory subcomplex containing ELP4, ELP5, and ELP6 (9,10). The copurification of the Elongator complex with the phosphorylated RNA polymerase II (RNAPII) (11) and the in vivo histone acetyl transferase (HAT) activity (12) suggested a role for the complex in RNAPII transcription elongation in yeast (11,12).…”

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confidence: 99%

Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation

Nelissen

Groeve

Fleury

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

114

192

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In eukaryotes, transcription of protein-encoding genes is strongly regulated by posttranslational modifications of histones that affect the accessibility of the DNA by RNA polymerase II (RNAPII). The Elongator complex was originally identified in yeast as a histone acetyltransferase (HAT) complex that activates RNAPII-mediated transcription. In Arabidopsis thaliana, the Elongator mutants elo1, elo2, and elo3 with decreased leaf and primary root growth due to reduced cell proliferation identified homologs of components of the yeast Elongator complex, Elp4, Elp1, and Elp3, respectively. Here we show that the Elongator complex was purified from plant cell cultures as a six-component complex. The role of plant Elongator in transcription elongation was supported by colocalization of the HAT enzyme, ELO3, with euchromatin and the phosphorylated form of RNAPII, and reduced histone H3 lysine 14 acetylation at the coding region of the SHORT HYPOCOTYL 2 auxin repressor and the LAX2 auxin influx carrier gene with reduced expression levels in the elo3 mutant. Additional auxin-related genes were down-regulated in the transcriptome of elo mutants but not targeted by the Elongator HAT activity showing specificity in target gene selection. Biological relevance was apparent by auxin-related phenotypes and marker gene analysis. Ethylene and jasmonic acid signaling and abiotic stress responses were up-regulated in the elo transcriptome and might contribute to the pleiotropic elo phenotype. Thus, although the structure of Elongator and its substrate are conserved, target gene selection has diverged, showing that auxin signaling and influx are under chromatin control.Arabidopsis | chromatin | histone acetyltranferase complex | SHY2

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“…The Elongator complex consists of six subunits, of which one displays HAT activity (4). Elongator copurifies with RNA polymerase II (RNAPII) during transcriptional elongation, presumably by rendering the DNA more accessible for the passage of the polymerase (5,6). In Arabidopsis thaliana (L.) Heyhn., 16 histone deacetylases and 12 HATs are encoded by the genome (7), and mutational analysis has already shown that some of these genes are involved in stress responses, growth, and flower development (8)(9)(10)(11).…”

mentioning

confidence: 99%

The elongata mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth

Nelissen

Fleury

Bruno

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

149

237

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The key enzyme for transcription of protein-encoding genes in eukaryotes is RNA polymerase II (RNAPII). The recruitment of this enzyme during transcription initiation and its passage along the template during transcription elongation is regulated through the association and dissociation of several complexes. Elongator is a histone acetyl transferase complex, consisting of six subunits (ELP1-ELP6), that copurifies with the elongating RNAPII in yeast and humans. We demonstrate that point mutations in three Arabidopsis thaliana genes, encoding homologs of the yeast Elongator subunits ELP1, ELP3 (histone acetyl transferase), and ELP4 are responsible for the phenotypes of the elongata2 (elo2), elo3, and elo1 mutants, respectively. The elo mutants are characterized by narrow leaves and reduced root growth that results from a decreased cell division rate. Morphological and molecular phenotypes show that the ELONGATA (ELO) genes function in the same biological process and the epistatic interactions between the ELO genes can be explained by the model of complex formation in yeast. Furthermore, the plant Elongator complex is genetically positioned in the process of RNAPII-mediated transcription downstream of Mediator. Our data indicate that the Elongator complex is evolutionarily conserved in structure and function but reveal that the mechanism by which it stimulates cell proliferation is different in yeast and plants.Arabidopsis ͉ histone acetyl transferase complex ͉ leaf development ͉ RNA polymerase II

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Purification and Characterization of the Human Elongator Complex

Cited by 234 publications

References 25 publications

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation

The elongata mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth

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