Periodic Reporting for period 3 - MaintainMeth (Quantitative analysis of DNA methylation maintenance within chromatin)
Période du rapport: 2020-04-01 au 2021-06-30
Cytosine methylation is a chemical modification that is precisely copied when DNA is replicated. Because methylation can regulate gene expression, accurate reproduction of DNA methylation patterns is essential for plant and animal development and for human health. The enzymes that maintain DNA methylation have to work within chromatin, and particularly to contend with nucleosomes – tight complexes of DNA and histone proteins. How methylation of nucleosomal DNA is maintained remains poorly understood. How methylation functions within chromatin, especially within active genes, has also been unclear and controversial.
My laboratory’s recent work with DDM1 – an ancient protein conserved between plants and animals that can move nucleosomes – and linker histone H1, which binds to nucleosomes and the intervening ‘linker’ DNA, has allowed us to formulate a hypothesis wherein movement of nucleosomes by DDM1 dislodges H1 and allows methyltransferases to access the DNA. My laboratory also discovered that DNA methylation influences nucleosome placement, thereby demonstrating that the interaction between DNA methylation and nucleosomes is bidirectional, and providing a possible mechanism through which DNA methylation can reguate gene activity.
Our goal is to deeply understand the connected processes of maintenance methylation and nucleosome placement, and how these affect gene function. This will be achieved through interconnected research strands: elucidation of how DNA methylation is maintained within chromatin, determination of how DNA methylation interacts with nucleosomes and H1 in vivo, and understanding of the functional consequences of DNA methylation within genes.
My laboratory’s recent work with DDM1 – an ancient protein conserved between plants and animals that can move nucleosomes – and linker histone H1, which binds to nucleosomes and the intervening ‘linker’ DNA, has allowed us to formulate a hypothesis wherein movement of nucleosomes by DDM1 dislodges H1 and allows methyltransferases to access the DNA. My laboratory also discovered that DNA methylation influences nucleosome placement, thereby demonstrating that the interaction between DNA methylation and nucleosomes is bidirectional, and providing a possible mechanism through which DNA methylation can reguate gene activity.
Our goal is to deeply understand the connected processes of maintenance methylation and nucleosome placement, and how these affect gene function. This will be achieved through interconnected research strands: elucidation of how DNA methylation is maintained within chromatin, determination of how DNA methylation interacts with nucleosomes and H1 in vivo, and understanding of the functional consequences of DNA methylation within genes.
The fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases has long remained unresolved. Our research showed that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis and mouse. We found that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we showed that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can likely evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.
DNA methylation and histone H1 are known to mediate transcriptional silencing of genes and transposable elements, but how they interact has been unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. We showed that H1 is enriched in methylated sequences, including genes, of Arabidopsis, yet this enrichment is independent of DNA methylation. We found that loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, we showed that H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. We also found that hypomethylation of genes activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.
DNA methylation and histone H1 are known to mediate transcriptional silencing of genes and transposable elements, but how they interact has been unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. We showed that H1 is enriched in methylated sequences, including genes, of Arabidopsis, yet this enrichment is independent of DNA methylation. We found that loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, we showed that H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. We also found that hypomethylation of genes activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.
Our research so far has revealed how DNA methylation is maintained within nucleosomes, elucidated the relationship between DNA methylation and histone H1, and revealed the function of gene body methylation. We expect our ultimate output will be the creation of a mathematical model of DNA methylation maintenance that will incorporate the bidirectional interactions between methylation and nucleosomes. This breakthrough will revolutionize research in the field by permitting the development of precise, quantitative hypotheses about the maintenance and function of DNA methylation within chromatin.