Loss of Elp1 in cerebellar granule cell progenitors models ataxia phenotype of Familial Dysautonomia.

Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits in sensory and autonomic neurons, neuronal defects in the central nervous system have also been described. Although ELP1 expression remains high in the normal developing and adult cerebellum, its role in cerebellar development is unknown. To explore the role of Elp1 in the cerebellum, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent as early as 7 days after birth, when Elp1cKO animals also had fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 in the developing cerebellum, and suggests that loss of Elp1 in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.

ZmELP1, an Elongator complex subunit, is required for the maintenance of histone acetylation and RNA Pol II phosphorylation in maize kernels.

The Elongator complex was originally identified as an interactor of hyperphosphorylated RNA polymerase II (RNAPII) in yeast and has histone acetyltransferase (HAT) activity. However, the genome-wide regulatory roles of Elongator on transcriptional elongation and histone acetylation remain unclear. We characterized a maize miniature seed mutant, mn7 and map-based cloning revealed that Mn7 encodes one of the subunits of the Elongator complex, ZmELP1. ZmELP1 deficiency causes marked reductions in the kernel size and weight. Molecular analyses showed that ZmELP1 interacts with ZmELP3, which is required for H3K14 acetylation (H3K14ac), and Elongator complex subunits interact with RNA polymerase II (RNAPII) C-terminal domain (CTD). Genome-wide analyses indicated that loss of ZmELP1 leads to a significant decrease in the deposition of H3K14ac and the CTD of phosphorylated RNAPII on Ser2 (Ser2P). These chromatin changes positively correlate with global transcriptomic changes. ZmELP1 mutation alters the expression of genes involved in transcriptional regulation and kernel development. We also showed that the decrease of Ser2P depends on the deposition of Elongator complex-mediated H3K14ac. Taken together, our results reveal an important role of ZmELP1 in the H3K14ac-dependent transcriptional elongation, which is critical for kernel development.

Wheat Elongator Subunit 4 Negatively Regulates Freezing Tolerance by Regulating Ethylene Accumulation.

Freezing stress is a major factor limiting production and geographical distribution of temperate crops. Elongator is a six subunit complex with histone acetyl-transferase activity and is involved in plant development and defense responses in Arabidopsis thaliana. However, it is unknown whether and how an elongator responds to freezing stress in plants. In this study, we found that wheat elongator subunit 4 (TaELP4) negatively regulates freezing tolerance through ethylene signaling. TaELP4 promoter contained cold response elements and was up-regulated in freezing stress. Subcellular localization showed that TaELP4 and AtELP4 localized in the cytoplasm and nucleus. Silencing of TaELP4 in wheat with BSMV-mediated VIGS approach significantly elevated tiller survival rate compared to control under freezing stress, but ectopic expression of TaELP4 in Arabidopsis increased leaf damage and survival rate compared with Col-0. Further results showed that TaELP4 positively regulated ACS2 and ACS6 transcripts, two main limiting enzymes in ethylene biosynthesis. The determination of ethylene content showed that TaELP4 overexpression resulted in more ethylene accumulated than Col-0 under freezing stress. Epigenetic research showed that histone H3K9/14ac levels significantly increased in coding/promoter regions of AtACS2 and AtACS6 in Arabidopsis. RT-qPCR assays showed that the EIN2/EIN3/EIL1-CBFs-COR pathway was regulated by TaELP4 under freezing stress. Taken together, our results suggest that TaELP4 negatively regulated plant responses to freezing stress via heightening histone acetylation levels of ACS2 and ACS6 and increasing their transcription and ethylene accumulation.

CURLED LATER1 encoding the largest subunit of the Elongator complex has a unique role in leaf development and meristem function in rice.

The Elongator complex, which is conserved in eukaryotes, has multiple roles in diverse organisms. In Arabidopsis thaliana, Elongator is shown to be involved in development, hormone action and environmental responses. However, except for Arabidopsis, our knowledge of its function is poor in plants. In this study, we initially carried out a genetic analysis to characterize a rice mutant with narrow and curled leaves, termed curled later1 (cur1). The cur1 mutant displayed a heteroblastic change, whereby the mutant leaf phenotype appeared specifically at a later adult phase of vegetative development. The shoot apical meristem (SAM) was small and the leaf initiation rate was low, suggesting that the activity of the SAM seemed to be partially reduced in cur1. We then revealed that CUR1 encodes a yeast ELP1-like protein, the largest subunit of Elongator. Furthermore, disruption of OsELP3 encoding the catalytic subunit of Elongator resulted in phenotypes similar to those of cur1, including the timing of the appearance of mutant phenotypes. Thus, Elongator activity seems to be specifically required for leaf development at the late vegetative phase. Transcriptome analysis showed that genes involved in protein quality control were highly upregulated in the cur1 shoot apex at the later vegetative phase, suggesting the restoration of impaired proteins probably produced by partial defects in translational control due to the loss of function of Elongator. The differences in the mutant phenotype and gene expression profile between CUR1 and its Arabidopsis ortholog suggest that Elongator has evolved to play a unique role in rice development.

Clinical and molecular findings in a Turkish family with an ultra-rare condition, ELP2-related neurodevelopmental disorder.

Elongator is a multi-subunit protein complex bearing six different protein subunits, Elp1 to -6, that are highly conserved among eukaryotes. Elp2 is the second major subunit of Elongator and, together with Elp1 and Elp3, form the catalytic core of this essential complex. Pathogenic variants that affect the structure and function of the Elongator complex may cause neurodevelopmental disorders. Here, we report on a new family with three children affected with a severe form of intellectual disability along with spastic tetraparesis, choreoathetosis, and self injury. Molecular genetic analyses reveal a homozygous missense variant in the ELP2 gene (NM_018255.4 (ELP2): c.1385G > A (p.Arg462Gln)), while in silico studies suggest a loss of electrostatic interactions that may contribute to the overall stability of the encoded protein. We also include a comparison of the patients with ELP2-related neurodevelopmental disorder to those previously reported in the literature. Apart from being affected with intellectual disability, we have extremely limited clinical knowledge about patients harboring ELP2 variants. Besides providing support to the causal role of p.Arg462Gln in ELP2-related neurodevelopmental disorder, we add self-injurious behavior to the clinical phenotypic repertoire of the disease.

SSD1 modifies phenotypes of Elongator mutants.

The translational decoding properties of tRNAs are influenced by post-transcriptional modification of nucleosides in their anticodon region. The Elongator complex promotes the first step in the formation of 5-methoxycarbonylmethyl (mcm5), 5-methoxycarbonylhydroxymethyl (mchm5), and 5-carbamoylmethyl (ncm5) groups on wobble uridine residues in eukaryotic cytosolic tRNAs. Elongator mutants in yeast, worms, plants, mice, and humans not only show a tRNA modification defect, but also a diverse range of additional phenotypes. Even though the phenotypes are almost certainly caused by the reduced functionality of the hypomodified tRNAs in translation, the basis for specific phenotypes is not well understood. Here, we discuss the recent finding that the phenotypes of Saccharomyces cerevisiae Elongator mutants are modulated by the genetic background. This background-effect is largely due to the allelic variation at the SSD1 locus, which encodes an mRNA-binding protein involved in post-transcriptional regulation of gene expression. A nonsense ssd1 allele is found in several wild-type laboratory strains and the presence of this allele aggravates the stress-induced phenotypes of Elongator mutants. Moreover, other phenotypes, such as the histone acetylation and telomeric gene silencing defects, are dependent on the mutant ssd1 allele. Thus, SSD1 is a genetic modifier of the phenotypes of Elongator-deficient yeast cells.

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development.

Transfer RNAs (tRNAs) are the most post-transcriptionally modified RNA species. Some of these modifications, especially the ones located in the anti-codon loop, are required for decoding capabilities of tRNAs. Such is the case for 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U), synthetized by the Elongator complex. Mutants for its sub-units display pleiotropic phenotypes. In this paper, we analyze the role of elp3 (Elongator catalytic sub-unit) in zebrafish development. We found that it is required for trunk development; elp3 knock-down animals presented diminished levels of mcm5s2U and sonic hedgehog (Shh) signaling activity. Activation of this pathway was sufficient to revert the phenotype caused by elp3 knockdown, indicating a functional relationship between Elongator and Shh through a yet unknown molecular mechanism.

[tRNA Wobble Base Modifications and Boric Acid Resistance in Yeast: Boron-Resistant Deletion Mutants Induce the General Amino Acid Control Mechanism and Activate Boron Efflux].

Boric acid is essential for plants and has many vital roles in animals and microorganisms. However, its high doses are toxic to all organisms. We previously screened yeast deletion collections to identify boric acid-resistant and susceptible mutants to identify genes that play a role in boron tolerance. Here, we analyzed boron resistant mutants (elplΔ, elp3Δ, elp6Δ, ncs2Δ, ncs6Δ and ktil2Δ) for their abilities to modulate the general amino acid control system (GAAC) and to induce boron efflux pump ATR1. The mutants analyzed in this study lack the genes that play roles in tRNA Wobble base modifications. We found that all of the boron resistant mutants activated Gcn4-dependent reporter gene activity and increased the transcript level of the ATR1 gene. Additionally, boron resistant cells accumulated less boric acid in their cytoplasm compared to the wild type cells upon boron exposure. Thus, our findings suggested that loss of wobble base modifications in tRNA leads to GAAC activation and ATR1 induction, which in turn reduced intracellular boron levels and caused boron resistance.

Dimerization of elongator protein 1 is essential for Elongator complex assembly.

The evolutionarily conserved Elongator complex, which is composed of six subunits elongator protein 1 (Elp1 to -6), plays vital roles in gene regulation. The molecular hallmark of familial dysautonomia (FD) is the splicing mutation of Elp1 [also known as IκB kinase complex-associated protein (IKAP)] in the nervous system that is believed to be the primary cause of the devastating symptoms of this disease. Here, we demonstrate that disease-related mutations in Elp1 affect Elongator assembly, and we have determined the structure of the C-terminal portion of human Elp1 (Elp1-CT), which is sufficient for full-length Elp1 dimerization, as well as the structure of the cognate dimerization domain of yeast Elp1 (yElp1-DD). Our study reveals that the formation of the Elp1 dimer contributes to its stability in vitro and in vivo and is required for the assembly of both the human and yeast Elongator complexes. Functional studies suggest that Elp1 dimerization is essential for yeast viability. Collectively, our results identify the evolutionarily conserved dimerization domain of Elp1 and suggest that the pathological mechanisms underlying the onset and progression of Elp1 mutation-related disease may result from impaired Elongator activities.

The Arabidopsis ELP3/ELO3 and ELP4/ELO1 genes enhance disease resistance in Fragaria vesca L.

BACKGROUND: Plant immune response is associated with a large-scale transcriptional reprogramming, which is regulated by numerous transcription regulators such as the Elongator complex. Elongator is a multitasking protein complex involved in diverse cellular processes, including histone modification, DNA methylation, and tRNA modification. In recent years, Elongator is emerging as a key regulator of plant immune responses. However, characterization of Elongator's function in plant immunity has been conducted only in the model plant Arabidopsis thaliana. It is thus unclear whether Elongator's role in plant immunity is conserved in higher plants. The objective of this study is to characterize transgenic woodland strawberry (Fragaria vesca L.) overexpressing the Arabidopsis Elongator (AtELP) genes, AtELP3 and AtELP4, and to determine whether F. vesca carries a functional Elongator complex. METHODS: Transgenic F. vesca and Arabidopsis plants were produced via Agrobacterium-mediated genetic transformation and characterized by morphology, PCR, real-time quantitative PCR, and disease resistance test. The Student's t test was used to analyze the data. RESULTS: Overexpression of AtELP3 and AtELP4 in F. vesca impacts plant growth and development and confers enhanced resistance to anthracnose crown rot, powdery mildew, and angular leaf spot, which are caused by the hemibiotrophic fungal pathogen Colletotrichum gloeosporioides, the obligate biotrophic fungal pathogen Podosphaera aphanis, and the hemibiotrophic bacterial pathogen Xanthomonas fragariae, respectively. Moreover, the F. vesca genome encodes all six Elongator subunits by single-copy genes with the exception of FvELP4, which is encoded by two homologous genes, FvELP4-1 and FvELP4-2. We show that FvELP4-1 complemented the Arabidopsis Atelp4/elo1-1 mutant, indicating that FvELP4 is biologically functional. CONCLUSIONS: This is the first report on overexpression of Elongator genes in plants. Our results indicate that the function of Elongator in plant immunity is most likely conserved in F. vesca and suggest that Elongator genes may hold potential for helping mitigate disease severity and reduce the use of fungicides in strawberry industry.