IC4R006-Epigenomic-2016-22110044

Project Title

• High-resolution mapping of open chromatin in the rice genome

The Background of This Project

• The identification and functional characterization of the regula- tory DNA elements is essential for understanding the regulation of gene expression in eukaryotic genomes. Although the genomes of an increasing number of eukaryotic species have been sequenced, genome-wide identification of regulatory DNA elements, such as that being done in the ENCODE project (The ENCODE Project Consortium 2007) and the Epigenomics Roadmap (Bernstein et al. 2010) in humans and in the modENCODE projects in Caenorhabditis elegans and Drosophila melanogaster (Gerstein et al. 2010; Roy et al. 2010), has been initiated only in few species. Active regulatory DNA elements, such as promoter and enhancers, in- teract with regulatory proteins. As a result, these regions are either free of nucleosomes or are under dynamic nucleosome modifications or displacements (Henikoff et al. 2009; Jin et al. 2009). Thus, active DNA elements are associated with open chromatin in higher eukaryotic genomes. One distinct characteristic of the genomic regions of open chromatin is a pronounced sensitivity to cleavage of endonuclease DNase I (Wu 1980; Keene et al. 1981; McGhee et al. 1981). Almost all active regulatory elements, in- cluding promoters, enhancers, suppressors, insulators, and locus control regions, have been shown to be marked by DNase I hypersensitive (DH) sites. (Gross and Garrard 1988).
• Rice (Oryza sativa) is the most important food crop in the world and has also been established as a model species for plant genome research. Rice provides one of the most accurately sequenced genomes from any multicellular eukaryotes (Goff et al. 2002; Matsumoto et al. 2005). Extensive genome-wide DNA methylation and histone modification data sets have recently been generated in rice (Feng et al. 2010; He et al. 2010; Yan et al. 2010; Zemach et al. 2010).
• In this project , the researchers describe high-resolution maps of DH sites in rice from both seedling and callus tissues. We report a number of novel features associated with rice DH sites, including their epigenetic modifica- tions, dynamic response to tissue culture, and association with genes that differentially expressed genes in seedling and callus tissues.

Plant Materials & Treatment

• Mixed leaf and stem tissues of 2-wk-old rice cultivar ‘‘Nipponbare’’ seedlings grown in a greenhouse were collected and ground into a fine powder in liquid nitrogen. The resulting powder was suspended in nuclear isolation buffer (NIB; 20 mM Tris-HCl, 50 mM EDTA, 5 mM Spermidine, 0.15 mM Spermine, 0.1% mercaptoethanol, 40% Glycerol at pH 7.5) and followed the standard protocol for nuclei isolation. Rice callus tissue was induced from sterilized

Nipponbare seeds in rice calli induction medium (NB basal medium plus vitamin, glutamine, proline, casein hydrolysate, sucrose, and phytogel as well as 3 mg/L 2,4-D at pH 5.8) under 28°C–29°C with dark conditions. Three-week-old calli were col- lected for nuclei isolation using the same method as for leaf tissue. The prepared nuclei pellet was suspended in RSB buffer (10 mM Tris at pH 7.4, 10 mM NaCl, 3 mM MgCl 2 ) for DNase I (Roche) digestion with increasing concentrations (0–4 units) for 10 min at 37°C.

Labs working on this Project

• Department of Horticulture, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA;
• Department of Plant and Microbial Biology, University of California–Berkeley, Berkeley, California 94720, USA;
• Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina 27708, USA

Corresponding Author

• Jiming Jiang (jjiang1@wisc.edu)