LIGHTER

Elongator role in light-induced gene expression – from upstream activators to gene targets and mechanism behind the regulation of RNAPII transcription elongation

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 Obiettivo del progetto (Objective)

'Elongator is a histone acetyltransferase (HAT) complex associated with RNA polymerase II (RNAPII) to facilitate transcription elongation. It consists of subunits Elp1-6 with Elp3 conferring HAT activity. Elongator is conserved in yeast, plants and humans. In humans, mutations in Elp genes cause neuronal diseases. In plants, Elongator is a positive regulator of cell proliferation during leaf and root growth. Elongator mutants have altered auxin signaling and responses to abscisic acid. Elongator regulates expression of specific genes, such as the light-regulated auxin response regulator IAA3/SHY2 and the clock regulator LHY during RNAPII transcription elongation. In addition, Elongator mutant seedlings show an altered light response and resemble triple phytochrome mutants. The working hypothesis is that Elongator acts as interface between phytochrome signaling and gene expression during transcription elongation to regulate processes such as the shade avoidance response. Different light qualities (low and high red/far red ratios) and seedlings of Elongator mutant or recombinant lines will be used to explore light-related Elongator target genes, pathways and interactors. In a first objective, light-regulated genome-wide targets and pathways of Elongator will be determined using microarrays, chromatin immunoprecipitation (ChIP-on-chip or ChIP-Seq) and iTRAQ proteome analysis. In a second objective, interactors of Elongator involved in light signaling and their target-specificity will be identified using tandem affinity purification, transient split-luciferase in vivo assay and ChIP-PCR. Genetic interactions between Elongator and light signaling mutants will also be investigated. In the third objective, the mechanism of Elongator action will be studied using ChIP-qPCR to check whether Elongator target genes contain abundantly stalled RNAPII at their 5’ region. Novel insight into chromatin-related regulation of gene expression during light activation will be obtained.'

Introduzione (Teaser)

Researchers are investigating a protein complex, Elongator, that plays a major role in plant response to light.

Descrizione progetto (Article)

Transcription is a vital process for the production of functional gene products such as proteins or RNA. RNA polymerase II (RNAP) drives the transcription process to initiate gene expression and convert DNA contents into RNA. Elongator associates with the RNAP enzyme to enable the elongation process in transcription by modifying histones. Histones are proteins that associate with DNA in the cell nucleus and help maintain the chromosome structure.

Although Elongator is a positive regulator of plant growth, little is known about the genes targeted by Elongator and its protein interactors. The EU-funded LIGHTER project investigated the role and interactions of Elongator during the transcription process in plants in response to different light conditions. The hypocotyl assay was used to assess the response to darkness, red, far-red and blue light conditions.

LIGHTER scientists tested and compared the seedlings of plants with Elongator-mutant strains to wild type plants using micro-arrays to analyse their transcriptome. Transcriptome represents all the RNA present inside a cell. The mutant seedlings were characterised based on their structure and form as well as response to light. The study of plants' development in light and dark conditions revealed those light-sensitive genes that are targeted by Elongator. Results also revealed how Elongator interacts with photoreceptors (such as the phytochromes A and B) along with the associated signalling pathways.

Tandem affinity purification (TAP) followed by mass spectrometry enabled the identification of 50 potential protein interactors of Elongator. These included RNA binding proteins, sub-units of the 26S proteasome and light-related proteins.

Project outcomes should enable the elucidation of epigenetic gene regulation in plants when exposed to different light conditions due to histone alterations by Elongator. Comprehending the role of Elongator and its interactions with proteins, photoreceptors and light-induced signalling pathways will help in the development of accurate models. It is hoped that fundamental processes like the circadian clock, photomorphogenesis, photosynthesis and leaf development can be adequately described in these models.