Final Report Summary - EMRES (Establishing the meiotic recombination-initiation epigenetic code in the yeast Saccharomyces cerevisiae)
The objective of this project was to systematically characterise post-translational histone modifications that correlate with the selection and activation of meiotic recombination initiation sites in the model organism S. cerevisiae. In other words, we wished to reveal a 'histone code' that specified the different stages of meiotic recombination, with a special emphasis on the process of DNA double-strand break (DSB) formation.
A number of deletion mutants were constructed so as to inactivate genes coding for different histone modifying enzymes. In this system the progression of the whole meiotic program - meiotic S phase, DSB and crossover formation, sporulation efficiency, spore viability - were extensively characterised using up-to-date molecular biological and genetic techniques and the distribution of one histone mark (histone H3K56ac) was determined on the genome-wide scale using the chromatin immunoprecipitation on microarray (ChIP-on-Chip) technique.
The results showed that all the tested histone-modifying enzyme mutants (with the exception of set1 deletion) had only mild meiotic defects: normal S phase progress, slightly delayed DSB and crossover formation, delayed meiotic divisions and fully viable spores were seen. Genome-wide mapping of H3K56ac is sporulating yeast cells revealed a stochastic distribution of this histone tag, and no spatial correlation was found between the sites of meiotic DSBs and H3K56ac. The limited throughput of the original experimental strategy (as to the number of histone modifications tested) prompted us to develop a more flexible system with high-throughput screening potential.
This unique histone-screen made is possible to systematically address the (direct) effects of a large number of histone modifications. Twenty-six histone mutations were tested and by the end of the project the whole meiotic program was comprehensively characterised in the mutant library. Five novel histone point-mutations were discovered that genetically affected the process of meiotic DSB formation at two independent recombination hotspots tested. DSB mapping were extended to the genome-wide scale in the relevant mutants using the chip-on-chip and the 'thoroughly modern' next generation sequencing technologies (chip-Seq). The effects of the discovered histone point mutations on the overall gene-expression profiles (that might indirectly affect meiotic DSB profiles) were also revealed by microarray-based transcriptome analyses of time-course meiotic samples.
Furthermore, in one mutant the genome-wide distribution of meiotic crossovers and gene conversions (the two outcomes of meiotic DSBs) were determined by next generation sequencing of meiotic segregants (in collaboration with Jennifer Fung's team; San Francisco, USA).
Concluding remarks: in this EU project an interdisciplinary approach was applied to decipher the epigenetic language of meiotic recombination. The methods involved 'classical' as well as 'next generation' technologies - producing novel observations and findings. However, data analysis - possibly the most challenging part of this 24 month-long project - have not come to an end yet (e.g. a single NGS run generated about 40 terabytes (!) of raw data; analysing 48 microarrays is also a demanding task by itself), but much progress is expected from close collaboration between the biologists and the informaticians involved. Notwithstanding this bottleneck, the experiments realised in this EU project have improved our knowledge on how programmed Spo11-dependent DSB formation is controlled in relation to chromatin context and coupled to homologous recombination, and we are convinced that in the very near future these findings will lead to new far-reaching avenues in the field of recombination and epigenetics.
A number of deletion mutants were constructed so as to inactivate genes coding for different histone modifying enzymes. In this system the progression of the whole meiotic program - meiotic S phase, DSB and crossover formation, sporulation efficiency, spore viability - were extensively characterised using up-to-date molecular biological and genetic techniques and the distribution of one histone mark (histone H3K56ac) was determined on the genome-wide scale using the chromatin immunoprecipitation on microarray (ChIP-on-Chip) technique.
The results showed that all the tested histone-modifying enzyme mutants (with the exception of set1 deletion) had only mild meiotic defects: normal S phase progress, slightly delayed DSB and crossover formation, delayed meiotic divisions and fully viable spores were seen. Genome-wide mapping of H3K56ac is sporulating yeast cells revealed a stochastic distribution of this histone tag, and no spatial correlation was found between the sites of meiotic DSBs and H3K56ac. The limited throughput of the original experimental strategy (as to the number of histone modifications tested) prompted us to develop a more flexible system with high-throughput screening potential.
This unique histone-screen made is possible to systematically address the (direct) effects of a large number of histone modifications. Twenty-six histone mutations were tested and by the end of the project the whole meiotic program was comprehensively characterised in the mutant library. Five novel histone point-mutations were discovered that genetically affected the process of meiotic DSB formation at two independent recombination hotspots tested. DSB mapping were extended to the genome-wide scale in the relevant mutants using the chip-on-chip and the 'thoroughly modern' next generation sequencing technologies (chip-Seq). The effects of the discovered histone point mutations on the overall gene-expression profiles (that might indirectly affect meiotic DSB profiles) were also revealed by microarray-based transcriptome analyses of time-course meiotic samples.
Furthermore, in one mutant the genome-wide distribution of meiotic crossovers and gene conversions (the two outcomes of meiotic DSBs) were determined by next generation sequencing of meiotic segregants (in collaboration with Jennifer Fung's team; San Francisco, USA).
Concluding remarks: in this EU project an interdisciplinary approach was applied to decipher the epigenetic language of meiotic recombination. The methods involved 'classical' as well as 'next generation' technologies - producing novel observations and findings. However, data analysis - possibly the most challenging part of this 24 month-long project - have not come to an end yet (e.g. a single NGS run generated about 40 terabytes (!) of raw data; analysing 48 microarrays is also a demanding task by itself), but much progress is expected from close collaboration between the biologists and the informaticians involved. Notwithstanding this bottleneck, the experiments realised in this EU project have improved our knowledge on how programmed Spo11-dependent DSB formation is controlled in relation to chromatin context and coupled to homologous recombination, and we are convinced that in the very near future these findings will lead to new far-reaching avenues in the field of recombination and epigenetics.