Final Report Summary - CHRODIGI (Chromatin Dynamics In Genome Integrity)
Introduction
Preserving genomic stability is essential for the viability of the cell. Genome instability and loss of genome integrity is a hallmark of cancer and strongly associated with cancer predisposition.
Maintenance of genome integrity is an intricate process, involving an extensive protein network and coordination of many cellular pathways. Moreover, constant crosstalk between the repair process and pathways regulating cellular homeostasis (including cell cycle progression, silencing of unproductive repair pathways and transcription) is as important for cell viability and proliferation as the actual repair process.
It has become apparent that the chromatin organization of DNA along with repair pathways, is critical for the maintenance of genomic stability. In recent years, a multitude of chromatin regulatory factors such as histone post-translational modifications, histone variants and ATP-dependent chromatin-remodeling enzymes of the SWI/SNF family have been shown to play crucial roles in two processes essential for the maintenance of genome integrity: the DNA damage repair pathway and complete, accurate chromosomal replication. Although biochemical roles have been assigned to many of these factors, both the context in which these activities promote maintenance of genome stability and their biological functions are far from being understood.
The aim of this project is to understand how chromatin is integrated into the genome stability network and to decipher the interconnections between chromatin regulators and genome stability factors. Using budding yeast as a model system, the primary research focuses on the function of two evolutionary conserved chromatin factors: (i) the ATP-dependent chromatin remodeling enzyme INO80 and (ii) the histone variant H2A.Z.
INO80 is a multi-protein complex that has been implicated in many DNA metabolic processes including gene transcription, DNA replication and DNA damage repair. H2A.Z is a conserved member of the H2A-type histone variants that has been implicated in multiple nuclear processes such as gene transcription, DNA repair and chromosome segregation, whilst also regulating mammalian development and embryonic stem cell differentiation. Both INO80 and H2A.Z have been linked to maintenance of genome stability, however their function in protecting the integrity of the genome is unclear.
Published Work and Accomplishments
During the course of the Marie Curie Fellowship, Dr. Papamichos-Chronakis demonstrated that recruitment of chromatin regulators at a DNA double strand break is regulated by cell cycle and not ΥH2A.Xphosphorylation (collaboration with C.Peterson USA; Bennet, Papamichos-Chronakis* and Peterson*, Nature Communications, 2013). The fellow found that efficient double-strand break recruitment of the INO80, SWR-C, NuA4, SWI/SNF and RSC enzymes is inhibited by the non-homologous end-joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, they found find no significant role for H2A.X phosphorylation in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of H2A.X phosphorylation. This work indicates that cell cycle position has a key role in DNA repair pathway choice and that recruitment of chromatin regulators is tightly coupled to homologous recombination. This work challenged a central model in the field and shed new light on how chromatin is integrated into the DNA damage response. The fellow also co-authored a review article on the role of chromatin in genome stability (Papamichos-Chronakis and Peterson, Nature Reviews Genetics, 2013).
The team of Dr. Papamichos-Chronakis discovered an unanticipated connection between INO80 and the nuclear protein degradation network. Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). The fellow reported that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. They found that INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. These results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome (Lafon et al., Molecular Cell 2015). This study is the first to implicate a chromatin remodeler in the Ubiquitin-Proteasome System, linking chromatin regulation to nuclear protein turnover. Given the biological significance and novelty of these findings, the fellow is now focusing his studies on the physical and functional interconnection between chromatin and the protein degradation network during DNA metabolism.
In parallel to his research activities, the fellow established in 2012 and he is running a Synthetic Genetic Array (SGA) platform for high throughput yeast genetic analysis in Institut Curie.
Preserving genomic stability is essential for the viability of the cell. Genome instability and loss of genome integrity is a hallmark of cancer and strongly associated with cancer predisposition.
Maintenance of genome integrity is an intricate process, involving an extensive protein network and coordination of many cellular pathways. Moreover, constant crosstalk between the repair process and pathways regulating cellular homeostasis (including cell cycle progression, silencing of unproductive repair pathways and transcription) is as important for cell viability and proliferation as the actual repair process.
It has become apparent that the chromatin organization of DNA along with repair pathways, is critical for the maintenance of genomic stability. In recent years, a multitude of chromatin regulatory factors such as histone post-translational modifications, histone variants and ATP-dependent chromatin-remodeling enzymes of the SWI/SNF family have been shown to play crucial roles in two processes essential for the maintenance of genome integrity: the DNA damage repair pathway and complete, accurate chromosomal replication. Although biochemical roles have been assigned to many of these factors, both the context in which these activities promote maintenance of genome stability and their biological functions are far from being understood.
The aim of this project is to understand how chromatin is integrated into the genome stability network and to decipher the interconnections between chromatin regulators and genome stability factors. Using budding yeast as a model system, the primary research focuses on the function of two evolutionary conserved chromatin factors: (i) the ATP-dependent chromatin remodeling enzyme INO80 and (ii) the histone variant H2A.Z.
INO80 is a multi-protein complex that has been implicated in many DNA metabolic processes including gene transcription, DNA replication and DNA damage repair. H2A.Z is a conserved member of the H2A-type histone variants that has been implicated in multiple nuclear processes such as gene transcription, DNA repair and chromosome segregation, whilst also regulating mammalian development and embryonic stem cell differentiation. Both INO80 and H2A.Z have been linked to maintenance of genome stability, however their function in protecting the integrity of the genome is unclear.
Published Work and Accomplishments
During the course of the Marie Curie Fellowship, Dr. Papamichos-Chronakis demonstrated that recruitment of chromatin regulators at a DNA double strand break is regulated by cell cycle and not ΥH2A.Xphosphorylation (collaboration with C.Peterson USA; Bennet, Papamichos-Chronakis* and Peterson*, Nature Communications, 2013). The fellow found that efficient double-strand break recruitment of the INO80, SWR-C, NuA4, SWI/SNF and RSC enzymes is inhibited by the non-homologous end-joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, they found find no significant role for H2A.X phosphorylation in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of H2A.X phosphorylation. This work indicates that cell cycle position has a key role in DNA repair pathway choice and that recruitment of chromatin regulators is tightly coupled to homologous recombination. This work challenged a central model in the field and shed new light on how chromatin is integrated into the DNA damage response. The fellow also co-authored a review article on the role of chromatin in genome stability (Papamichos-Chronakis and Peterson, Nature Reviews Genetics, 2013).
The team of Dr. Papamichos-Chronakis discovered an unanticipated connection between INO80 and the nuclear protein degradation network. Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). The fellow reported that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. They found that INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. These results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome (Lafon et al., Molecular Cell 2015). This study is the first to implicate a chromatin remodeler in the Ubiquitin-Proteasome System, linking chromatin regulation to nuclear protein turnover. Given the biological significance and novelty of these findings, the fellow is now focusing his studies on the physical and functional interconnection between chromatin and the protein degradation network during DNA metabolism.
In parallel to his research activities, the fellow established in 2012 and he is running a Synthetic Genetic Array (SGA) platform for high throughput yeast genetic analysis in Institut Curie.