Final Report Summary - SG1_EPI_VAR (Functional characterization of Shoot Growth 1, a novel QTL-based epiallele)
An executive summary
Natural variation is widely used as a source of biodiversity to identify alleles controlling various traits. In the Institut Jean Pierre Bourgin (IJPB, INRA, Versailles) several groups are interested in the analysis of Arabidopsis natural variation. Classical approaches involving sets of Recombinant Inbred Lines (RIL) were developed and the genes explaining the QTLs were identified by map-based cloning and/or candidate gene approach. Interestingly, these very classical approaches led to the discovery of natural epialleles such as epiQTLs. The objective of the present project was to use my experience in the analysis of molecular mechanisms underlying naturally varying traits to study one of the very first examples of epiQTL allele isolated from crosses between natural populations of Arabidopsis. This project builds upon the work of Dr Daniela Vlad, a former PhD student in Dr Olivier Loudet's lab, who identified a novel natural epiallele named SG1. The proposed project focused on a further characterisation of this particular epiallele and of the underlying gene function.
The specific aims of this project was to:
- understand how variation at SG1 results in a dramatic effect on fitness;
- characterise the epigenetic status of the SG1 locus and its impact on the genome to gain a better understanding of the interplay between different epigenetic mechanisms leading to the regulation of plant growth.
The results from this study are likely to provide important insights into the regulation of gene expression controlled by DNA methylation, and are also likely to improve our understanding of the mechanisms by which plants integrate internal cues to adjust growth.
A summary description of project context and objectives
This research follows the discovery of QTL for leaf growth named Shoot growth1 (SG1), mapped in a population of recombinant inbred lines (RIL) resulting from the cross Bur-0 x Col-0. The observed phenotype is defects in leaf growth due to the presence of the allele in Col QTL in the homozygous state. Fine mapping have shown that SG1 corresponds to a single gene (unknown function, which we call SG1) through the analysis of recombinants from isogenic type heterogeneous inbred families (HIF) that limit the candidate region to 8 kb. A first study on SG1 showed that the gene is more methylated in SG1 in the Col accession compared to the Bur or Ct accessions, and several characteristics of the phenotype suggest that SG1 could have a function in the epigenetic regulation of multiple targets. The purpose of this project was to determine the role of SG1 and to study the protein in a functional way. Different approaches of genetics, biochemistry, molecular biology and cell biology were used to determine the cause of reduced growth phenotype resulting from the Col SG1 allele.
A description of the main science and technology (S&T) results / foregrounds
During the first period, several major results were obtained. First, we determined the cause of the phenotype associated with the SG1 allele in the Col genetic background. An analysis of the transcripts showed that a spontaneous mutation in an exon of the Col SG1 parent used in the cross Col x Bur is likely to be responsible for the growth defects. Indeed, this mutation converts a glutamine codon to a STOP, which potentially leads to the production of a truncated protein without the C-terminal domain. The SG1 allele containing the mutation in the Col parent named Q513STOP is being characterised and this phenotype is very similar to the one exhibited by the T-DNA mutants that were isolated in the SG1 gene. To gain insights into SG1 function, crosses were performed with various alleles involved in the processing of RNA-directed DNA methylation (RdDM) or DNA methylation at any cytosine context and allowed to place SG1 in an already known genetic pathway (undisclosed data).
In addition, a transcriptome analysis was performed and resulted in the identification of candidates that show for most of them an increased expression in the sg1 mutant background. These data have been compared to previous transcriptome data (CATMA) acquired from the HIF populations and multiple targets were subsequently been confirmed by quantitative PCR. It is worth noting that the majority of SG1 targets are involved in stress responses, including jasmonate response pathway (which has a known impact on growth).
We next performed a sg1 methylome compared to Col WT using high-throughput deep sequencing. The sequencing was performed by BGI, Beijin, and was analysed in collaboration with Dr Francisco Cubillos in the laboratory. Among main results, we observed an increase in cytosine methylation at several target genes that is likely to be associated with the phenotype confirming a role for SG1 in the epigenome regulation.
Concomitantly to DNA methylation, we explored the methylation status of two post-translational modifications at the histone H3 tail that are commonly associated with DNA methylation alterations as described above. The results obtained from these chromatin-immunoprecipitation experiments confirmed the potential function of SG1, and all these data are currently being written in a scientific article that will be submitted to a high impact peer-reviewed journal.
Concerning the analysis of SG1 protein function, we developed anti-peptide antibodies (Eurogentec) directed against the SG1 protein. These antibodies recognised efficiently the recombinant SG1 protein but failed to recognise native SG1 in total plant extracts or nuclear extract. In addition, we were not able to perform successful immunolocalisation experiments using these antibodies (conventional and confocal microscopy), despite several conditions tested. Therefore, these antibodies may only be suitable for in-vitro assays such as protein pulldowns. In parallel, the genomic sequence of SG1 was cloned and fused to GFP and HIS tags with expression driven by the native SG1 promoter (2.5 kb), but we were not able to detect a signal in western blot on plant extracts for both constructs, or to visualise GFP for the former fusion. The protein characterisation will be pursued in the lab by a PhD student using different strategies including over-expression given the very low abundance of SG1.
The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
Through this work, we identified and characterised a novel player in the epigenome regulation, of significant interest for the epigenetics community. This collaborative work performed in the laboratory of Dr Olivier Loudet and Dr Nicolas Bouché should lead to the publication of a high-quality article in a peer-reviewed journal during the year 2013, currently under writing. Data from this study will be largely presented at international conferences once the manuscript has been published.
Natural variation is widely used as a source of biodiversity to identify alleles controlling various traits. In the Institut Jean Pierre Bourgin (IJPB, INRA, Versailles) several groups are interested in the analysis of Arabidopsis natural variation. Classical approaches involving sets of Recombinant Inbred Lines (RIL) were developed and the genes explaining the QTLs were identified by map-based cloning and/or candidate gene approach. Interestingly, these very classical approaches led to the discovery of natural epialleles such as epiQTLs. The objective of the present project was to use my experience in the analysis of molecular mechanisms underlying naturally varying traits to study one of the very first examples of epiQTL allele isolated from crosses between natural populations of Arabidopsis. This project builds upon the work of Dr Daniela Vlad, a former PhD student in Dr Olivier Loudet's lab, who identified a novel natural epiallele named SG1. The proposed project focused on a further characterisation of this particular epiallele and of the underlying gene function.
The specific aims of this project was to:
- understand how variation at SG1 results in a dramatic effect on fitness;
- characterise the epigenetic status of the SG1 locus and its impact on the genome to gain a better understanding of the interplay between different epigenetic mechanisms leading to the regulation of plant growth.
The results from this study are likely to provide important insights into the regulation of gene expression controlled by DNA methylation, and are also likely to improve our understanding of the mechanisms by which plants integrate internal cues to adjust growth.
A summary description of project context and objectives
This research follows the discovery of QTL for leaf growth named Shoot growth1 (SG1), mapped in a population of recombinant inbred lines (RIL) resulting from the cross Bur-0 x Col-0. The observed phenotype is defects in leaf growth due to the presence of the allele in Col QTL in the homozygous state. Fine mapping have shown that SG1 corresponds to a single gene (unknown function, which we call SG1) through the analysis of recombinants from isogenic type heterogeneous inbred families (HIF) that limit the candidate region to 8 kb. A first study on SG1 showed that the gene is more methylated in SG1 in the Col accession compared to the Bur or Ct accessions, and several characteristics of the phenotype suggest that SG1 could have a function in the epigenetic regulation of multiple targets. The purpose of this project was to determine the role of SG1 and to study the protein in a functional way. Different approaches of genetics, biochemistry, molecular biology and cell biology were used to determine the cause of reduced growth phenotype resulting from the Col SG1 allele.
A description of the main science and technology (S&T) results / foregrounds
During the first period, several major results were obtained. First, we determined the cause of the phenotype associated with the SG1 allele in the Col genetic background. An analysis of the transcripts showed that a spontaneous mutation in an exon of the Col SG1 parent used in the cross Col x Bur is likely to be responsible for the growth defects. Indeed, this mutation converts a glutamine codon to a STOP, which potentially leads to the production of a truncated protein without the C-terminal domain. The SG1 allele containing the mutation in the Col parent named Q513STOP is being characterised and this phenotype is very similar to the one exhibited by the T-DNA mutants that were isolated in the SG1 gene. To gain insights into SG1 function, crosses were performed with various alleles involved in the processing of RNA-directed DNA methylation (RdDM) or DNA methylation at any cytosine context and allowed to place SG1 in an already known genetic pathway (undisclosed data).
In addition, a transcriptome analysis was performed and resulted in the identification of candidates that show for most of them an increased expression in the sg1 mutant background. These data have been compared to previous transcriptome data (CATMA) acquired from the HIF populations and multiple targets were subsequently been confirmed by quantitative PCR. It is worth noting that the majority of SG1 targets are involved in stress responses, including jasmonate response pathway (which has a known impact on growth).
We next performed a sg1 methylome compared to Col WT using high-throughput deep sequencing. The sequencing was performed by BGI, Beijin, and was analysed in collaboration with Dr Francisco Cubillos in the laboratory. Among main results, we observed an increase in cytosine methylation at several target genes that is likely to be associated with the phenotype confirming a role for SG1 in the epigenome regulation.
Concomitantly to DNA methylation, we explored the methylation status of two post-translational modifications at the histone H3 tail that are commonly associated with DNA methylation alterations as described above. The results obtained from these chromatin-immunoprecipitation experiments confirmed the potential function of SG1, and all these data are currently being written in a scientific article that will be submitted to a high impact peer-reviewed journal.
Concerning the analysis of SG1 protein function, we developed anti-peptide antibodies (Eurogentec) directed against the SG1 protein. These antibodies recognised efficiently the recombinant SG1 protein but failed to recognise native SG1 in total plant extracts or nuclear extract. In addition, we were not able to perform successful immunolocalisation experiments using these antibodies (conventional and confocal microscopy), despite several conditions tested. Therefore, these antibodies may only be suitable for in-vitro assays such as protein pulldowns. In parallel, the genomic sequence of SG1 was cloned and fused to GFP and HIS tags with expression driven by the native SG1 promoter (2.5 kb), but we were not able to detect a signal in western blot on plant extracts for both constructs, or to visualise GFP for the former fusion. The protein characterisation will be pursued in the lab by a PhD student using different strategies including over-expression given the very low abundance of SG1.
The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
Through this work, we identified and characterised a novel player in the epigenome regulation, of significant interest for the epigenetics community. This collaborative work performed in the laboratory of Dr Olivier Loudet and Dr Nicolas Bouché should lead to the publication of a high-quality article in a peer-reviewed journal during the year 2013, currently under writing. Data from this study will be largely presented at international conferences once the manuscript has been published.