Lanterna Rally

Author: Min Kim

Publisher:

Published: 2013

Total Pages: 99

ISBN-13:

Chromatin is a mixture of DNA and DNA binding proteins that control transcription. Dynamic chromatin structure modulates gene expression and is responsible for an extraordinary spectrum of developmental processes. An intricate interplay of DNA methylation, histone modifications, histone variants, small RNA accumulation, and ATPase chromatin remodelers defines chromatin re-configuration in a precise manner, locally within a cell and globally across different cell types. The development of high-throughput screening methods such as microarray and whole-genome sequencing has led to an explosion of chromatin studies in the past decade. Moreover, genetic and molecular studies resulted in identification of a number of proteins that may influence chromatin structure. However, the exact functions of individual proteins as well as their functional relationships to each other are less understood. Also, the role of chromatin components in establishing cell- and tissue-specific chromatin structure is largely unknown. To address these open questions in chromatin biology, I focused my dissertation work on 1) studying tissue-specific DNA demethylation in seed, and 2) determining the role of a ubiquitous DNA binding protein, linker histone H1, in regulating chromatin structure. Tissue specific DNA methylation in seed. In endosperm, the nutritive tissue that nourishes the embryo, parent-of-origin specific gene expression is regulated by DNA demethylation. However, the extent to which DNA demethylation occurs in a tissue-specific manner and regulates transcription in the endosperm of crop plants like rice remains unknown. To address these questions, my colleagues and I examined the DNA methylation patterns of two rice seed tissues, embryo and endosperm. We found that endosperm genome is globally hypomethylated at non-CG sites and locally hypomethylated at CG-sites compared to embryo. We also identified that small transposons near genes (euchromatic regions) are the primary targets of DNA demethylation. The loci near the genes preferentially expressed in endosperm (e.g. storage protein and starch synthesizing enzymes) are subjected to local hypomethylation, suggesting that DNA methylation plays a role in inducing tissue-specific genes in endosperm. The role of H1 in regulating chromatin structure. H1 is proposed to facilitate higher order chromatin structure, but its effects on individual chromatin components and transcription are less understood. To resolve this issue, we investigated the role of H1 in regulating DNA methylation, nucleosome positioning, and transcription. We identified that H1 was most enriched in transposons. H1 was also found in genes at a lower level compared to transposons, and the abundance of H1 was anticorrelated with gene expression. Moreover, H1 influences nucleosome positioning by increasing the distance between two nucleosomes. Lack of H1 resulted in increased DNA methylation of transposons with heterochromatic features. In contrast, an h1 mutant showed a reduction of DNA methylation in genes and transposons with euchromatic features. Our finding suggests that H1 has a dual function in regulating DNA methylation. That is, H1 inhibits both DNA methyltransferases and DNA demethylation-associated enzymes from binding heterochromatin and euchromatin, respectively. In addition, the hypermethylated loci in our h1 mutant almost perfectly overlapped with the hypomethylated loci in a ddm1 mutant in heterochromatin, suggesting a link between these two proteins. DDM1 is an Snf2 chromatin remodeler that can slide nucleosomes along DNA and has been proposed to provide DNA methyltransferase access to target sequences. We further determined their functional relationship by crossing h1 and ddm1 mutants, and generated a map of DNA methylation of the cross. We identified that loss of DNA methylation from ddm1 was partially recovered by removing H1. Also the mutant phenotype observed in ddm1 disappeared in h1ddm1. Based on our results, we proposed a model where DDM1-mediated chromatin destabilization releases H1 binding, which in turn increases DNA accessibility. It is noteworthy that DNA demethylation preferentially occurred in euchromatin in both the rice seed DNA methylation study and the H1 study. Based on this result, we proposed that the apparent target preference of DNA demethylation-associated proteins depends on the underlying chromatin structure. We think that this chromatin structure-mediated specificity also dictates other nucleoproteins to determine/recognize their targets. My dissertation work tackled multiple aspects of chromatin biology: tissue-specific chromatin regulation, and the interplay between chromatin components in chromatin organization. Together, the results from my work enhanced our knowledge of how chromatin components influence overall chromatin structure.