Final Report Summary - LIGHTER (Elongator role in light-induced gene expression - from upstream activators to gene targets and mechanism behind the regulation of RNAPII transcription elongation)
Expression of genes starts with transcription of message encoded in deoxyribonucleic acid (DNA) into ribonucleic acid (RNA) molecule. Transcription of genes encoding proteins is driven by the enzyme named RNA polymerase II. This enzyme needs factors which facilitate initiation or elongation of transcription. Elongator is a protein complex which associates with RNA polymerase II to facilitate elongation of transcription by modification of histones (proteins interacting with DNA). Elongator was discovered in yeast, plants and humans. Mutations in human Elongator cause neuronal diseases. In plants, Elongator is a positive regulator of leaf and root growth but stll we know only very few genes targeted by Elongator (transcription of which is facilitated by Elongator).
The main aim of this project was to show that Elongator acts as interface between light signalling and gene expression during transcription elongation. Our objectives were to identify light-related Elongator target genes and protein interactors.
We analysed how mutations or overexpression of genes encoding subunits of the Elongator complex affect response to red, far-red and blue light. We applied the method called hypocotyl assay - the seedlings of mutants and overexpressors were grown in the given light qualities and their length was compared to the wild type plants. We also performed two microarray experiments to compare transcriptomes (all transcribed genes) of the Elongator mutant and the wild type plants grown in darkness or grown in darkness and illuminated with red or far-red light. We obtained seven double or triple mutants combining the mutation in Elongator and mutations in photoreceptors phytochrome A, phytochrome B or other genes coding for components of light signalling pathways. The mutants were characterised with respect to their morphology and response to red, far-red and blue light. To identify protein interactors of Elongator the tandem affinity purification (TAP) was performed. In this approach, Elongator was purified from seedlings together with interacting proteins which were identified by mass spectrometry.
Based on the results of hypocotyl assay, we concluded that he whole Elongator complex takes part in the light response to red, far-red and blue light, respectively. The double and triple mutant analysis indicated that Elongator interacts genetically with phyA, functions in the same pathway with phyB, and it contributes to response to far-red light but independently from HFR1 - transcription factor and positive regulator of photomotphogenesis (plant development in light). Putative target genes of Elongator were identified by microarrays: genes of circadian clock components, involved in photo- or skotomorphogenesis (plant development in darkness), related to chloroplasts biogenesis and chlorophyll biosynthesis, genes coding for subunits of light harvesting complex or for transcription factors regulating leaf development. Some of those genes are targeted both in darkness and after the short red and/or far-red light treatment while others seem to be light-specific targets of Elongator. The TAP method identified 50 potential interactors of Elongator including three most interesting classes: RNA binding proteins, subunits of the regulatory particle of the 26S proteasome and light related proteins.
Identification of genes targeted by Elongator will allow to define the set of biological processes regulated by this factor at the epigenetic level. Discovery of genetic interactions between Elongator and photoreceptors and other light signalling components will clarify the position of Elongator within the network of light induced pathways. Identification of proteins interacting with Elongator should explain how the light signal is transmitted to this protein complex and how Elongator is guided to target genes. We expect that as the final result of this project we can propose the model in which Elongator will link phyA and phyB - two main plant light receptors, and fundamental processes like circadian clock, photomorphogenesis, photosynthesis and leaf development. Importantly, since Elongator regulates gene expression via histone modification, our findings will contribute to intensely investigated field of epigenetic gene regulation in response to light.
The main aim of this project was to show that Elongator acts as interface between light signalling and gene expression during transcription elongation. Our objectives were to identify light-related Elongator target genes and protein interactors.
We analysed how mutations or overexpression of genes encoding subunits of the Elongator complex affect response to red, far-red and blue light. We applied the method called hypocotyl assay - the seedlings of mutants and overexpressors were grown in the given light qualities and their length was compared to the wild type plants. We also performed two microarray experiments to compare transcriptomes (all transcribed genes) of the Elongator mutant and the wild type plants grown in darkness or grown in darkness and illuminated with red or far-red light. We obtained seven double or triple mutants combining the mutation in Elongator and mutations in photoreceptors phytochrome A, phytochrome B or other genes coding for components of light signalling pathways. The mutants were characterised with respect to their morphology and response to red, far-red and blue light. To identify protein interactors of Elongator the tandem affinity purification (TAP) was performed. In this approach, Elongator was purified from seedlings together with interacting proteins which were identified by mass spectrometry.
Based on the results of hypocotyl assay, we concluded that he whole Elongator complex takes part in the light response to red, far-red and blue light, respectively. The double and triple mutant analysis indicated that Elongator interacts genetically with phyA, functions in the same pathway with phyB, and it contributes to response to far-red light but independently from HFR1 - transcription factor and positive regulator of photomotphogenesis (plant development in light). Putative target genes of Elongator were identified by microarrays: genes of circadian clock components, involved in photo- or skotomorphogenesis (plant development in darkness), related to chloroplasts biogenesis and chlorophyll biosynthesis, genes coding for subunits of light harvesting complex or for transcription factors regulating leaf development. Some of those genes are targeted both in darkness and after the short red and/or far-red light treatment while others seem to be light-specific targets of Elongator. The TAP method identified 50 potential interactors of Elongator including three most interesting classes: RNA binding proteins, subunits of the regulatory particle of the 26S proteasome and light related proteins.
Identification of genes targeted by Elongator will allow to define the set of biological processes regulated by this factor at the epigenetic level. Discovery of genetic interactions between Elongator and photoreceptors and other light signalling components will clarify the position of Elongator within the network of light induced pathways. Identification of proteins interacting with Elongator should explain how the light signal is transmitted to this protein complex and how Elongator is guided to target genes. We expect that as the final result of this project we can propose the model in which Elongator will link phyA and phyB - two main plant light receptors, and fundamental processes like circadian clock, photomorphogenesis, photosynthesis and leaf development. Importantly, since Elongator regulates gene expression via histone modification, our findings will contribute to intensely investigated field of epigenetic gene regulation in response to light.