Identification and Characterization of Factors Involved in Active DNA Demethylation and Epigenetic Anti-Silencing in Arabidopsis thaliana
DNA methylation is a conserved epigenetic modification involved in the regulation of gene expression. A proper DNA methylation level is critical for many physiological and developmental processes in animals and plants. As some mechanisms by which DNA methylation is regulated are conserved, studies of how DNA methylation is controlled in plants can improve our understanding of the epigenetic regulation of gene expression in both plants and animals. Active DNA demethylation plays critical roles in shaping the genome-wide DNA methylation pattern. In my thesis research concerning active DNA demethylation and other anti-silencing mechanisms, a transgene-based forward genetic screen was used to identify several new anti-silencing factors in Arabidopsis . The functions of these new anti-silencing factors in active DNA demethylation and transcriptional anti-silencing were characterized. In chapter 2, the characterization of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein in Arabidopsis , is described. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. ASI1 ensures the proper expression of IBM1 full-length transcript. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3′ distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements. Chapter 3 describes the characterization of an anti-silencing factor, ASI3/MET18, the dysfunction of which causes transgene promoter hyper-methylation and silencing. MET18 is a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Our study shows that ROS1 physically associates with MET18 and other CIA components. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. Chapter 4 describes the identification and characterization of a pair of Harbinger transposon-derived proteins (HDPs), HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDM1, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. Our study revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing.
Zhu, Purdue University.
Molecular biology|Genetics|Plant sciences
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