Final Report Summary - SRA AND EPIGENETICS (The SRA domain: a connection between DNA methylation and histone modifications within the epigenetic code? The Arabidopsis VIM family)
Project objectives
In this proposal, I planned to characterise the role of the arabidopsis SRA-domain containing proteins family (vimentin (VIM) family) in deoxyribonucleic acid (DNA) methylation control, and how this is related with histone acetylation and methylation in a way to regulate gene transcription and the epigenetic code. However, just after submitting the application for this grant, a paper was published describing the function and potential redundancy among the VIM proteins, where they investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes (Woo et al., 2008). They showed that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.
The publication of the paper by the Richards lab (Woo et al., 2008) made us change the work plan completely and develop a new plan, also of high interest in the field of epigenetics, as explained in the midterm report and first period periodic report. The revised objectives of the project have been:
- in depth analysis of the replication-related phenotype of atxr5/6 mutants and their relationship with other heterochromatin-related factors;
- study of heterochromatin formation.
Work since the beginning of the project and main results achieved
Due to all this published work from the Richards lab, I decided to focus my efforts in pursuing different aims:
- Study of ATXR5/6 function in DNA replication: my expertise in the field of DNA replication proved very useful for my co-workers and we were able to publish a paper in Nature on how histone methyl transferase ATXR5 and six mutants show localised re-replication in heterochromatin (collaboration between the outgoing and return phases laboratories in the United States (US) and Europe) (Jacob Y., Stroud H., Leblanc C., Feng S., Zhuo L., Caro E., Hassel C., Gutierrez C., Michaels S. D., Jacobsen S. E., Nature. 2010). A follow up manuscript was later published where we showed that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, ribonucleic acid (RNA) sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs) (Stroud H., Hale C. J., Feng S., Caro E., Jacob Y., Michaels S. D., Jacobsen S. E. PLoS Genet. 2012).
- Study of DNA heterochromatin formation: Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals it's generally characterised by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually co-located and set up a mutually self-reinforcing and stable state. In the last year, the study of SUVR5 function and mechanism of action became my main project, and I was able to find that this protein, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues. (Caro E., Stroud H., Greenberg M. V., Bernatavichute Y. V., Feng S., Groth M., Vashisht A. A., Wohlschlegel J., Jacobsen S. E., PLoS Genet. 2012).
During 2012, I also became involved in the study of the relationship between H3K9me2 and DNA methylation. CHG methylation by Chromomethylase3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. We were able to show multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2, and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (BAH) and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains and reveals a distinct mechanism of interplay between DNA methylation and histone modification (Du J., Zhong X., Bernatavichute Y. V., Stroud H., Feng S., Caro E., Vashisht A. A., Terragni J., Chin H. G., Tu A., Hetzel J., Wohlschlegel J. A., Pradhan S., Patel D. J., Jacobsen S. E., Cell. 2012).
Transfer of knowledge:
The work on SUVR5 and its role as a targetable gene silencer and expression repression was considered interesting enough by University of California, Los Angeles (UCLA) to file a US provisional patent application (serial No. 61/585,619. Methods and compositions for reducing gene expression in plants. Named inventors: Steven E. Jacobsen and Elena Caro Bernat). We are now working on converting this provisional patent into a PTO.
During all these two years in the Jacobsen lab, I have worked closely with three undergraduate students from UCLA that became very successfully trained in molecular biology techniques (Christopher Blair, now in graduate school in UC Irvine, Shui Ho Chan, now in medical school in Hong Kong, and Ashley Gomez, now graduated and applying for Medical School).
Expected final results and potential impact and use
The final results from the project have all been already published and they will probably have a high impact on the epigenetics field since they were all published in first levels journals.
The filing of the patent on SUVR5 makes it susceptible of being really used to produce an impact in a wider societal level.
In this proposal, I planned to characterise the role of the arabidopsis SRA-domain containing proteins family (vimentin (VIM) family) in deoxyribonucleic acid (DNA) methylation control, and how this is related with histone acetylation and methylation in a way to regulate gene transcription and the epigenetic code. However, just after submitting the application for this grant, a paper was published describing the function and potential redundancy among the VIM proteins, where they investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes (Woo et al., 2008). They showed that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.
The publication of the paper by the Richards lab (Woo et al., 2008) made us change the work plan completely and develop a new plan, also of high interest in the field of epigenetics, as explained in the midterm report and first period periodic report. The revised objectives of the project have been:
- in depth analysis of the replication-related phenotype of atxr5/6 mutants and their relationship with other heterochromatin-related factors;
- study of heterochromatin formation.
Work since the beginning of the project and main results achieved
Due to all this published work from the Richards lab, I decided to focus my efforts in pursuing different aims:
- Study of ATXR5/6 function in DNA replication: my expertise in the field of DNA replication proved very useful for my co-workers and we were able to publish a paper in Nature on how histone methyl transferase ATXR5 and six mutants show localised re-replication in heterochromatin (collaboration between the outgoing and return phases laboratories in the United States (US) and Europe) (Jacob Y., Stroud H., Leblanc C., Feng S., Zhuo L., Caro E., Hassel C., Gutierrez C., Michaels S. D., Jacobsen S. E., Nature. 2010). A follow up manuscript was later published where we showed that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, ribonucleic acid (RNA) sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs) (Stroud H., Hale C. J., Feng S., Caro E., Jacob Y., Michaels S. D., Jacobsen S. E. PLoS Genet. 2012).
- Study of DNA heterochromatin formation: Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals it's generally characterised by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually co-located and set up a mutually self-reinforcing and stable state. In the last year, the study of SUVR5 function and mechanism of action became my main project, and I was able to find that this protein, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues. (Caro E., Stroud H., Greenberg M. V., Bernatavichute Y. V., Feng S., Groth M., Vashisht A. A., Wohlschlegel J., Jacobsen S. E., PLoS Genet. 2012).
During 2012, I also became involved in the study of the relationship between H3K9me2 and DNA methylation. CHG methylation by Chromomethylase3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. We were able to show multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2, and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (BAH) and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains and reveals a distinct mechanism of interplay between DNA methylation and histone modification (Du J., Zhong X., Bernatavichute Y. V., Stroud H., Feng S., Caro E., Vashisht A. A., Terragni J., Chin H. G., Tu A., Hetzel J., Wohlschlegel J. A., Pradhan S., Patel D. J., Jacobsen S. E., Cell. 2012).
Transfer of knowledge:
The work on SUVR5 and its role as a targetable gene silencer and expression repression was considered interesting enough by University of California, Los Angeles (UCLA) to file a US provisional patent application (serial No. 61/585,619. Methods and compositions for reducing gene expression in plants. Named inventors: Steven E. Jacobsen and Elena Caro Bernat). We are now working on converting this provisional patent into a PTO.
During all these two years in the Jacobsen lab, I have worked closely with three undergraduate students from UCLA that became very successfully trained in molecular biology techniques (Christopher Blair, now in graduate school in UC Irvine, Shui Ho Chan, now in medical school in Hong Kong, and Ashley Gomez, now graduated and applying for Medical School).
Expected final results and potential impact and use
The final results from the project have all been already published and they will probably have a high impact on the epigenetics field since they were all published in first levels journals.
The filing of the patent on SUVR5 makes it susceptible of being really used to produce an impact in a wider societal level.