,Integration of Cytological Features with Molecular and Epigenetic Properties of Rice Chromosome 4

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It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-leveI transcriptiOnaI activity.To better understand this chromosomaI organization,we created a modeI based on the statisticaI distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining.Fluorescence in.situ hvbridization(FISH)experiments using a set of bacteriaI artificiaI chromosome(BAC)clones from chromosome 4 placed all 18 clones in the region predicted by the model.Elevated levels of H3K4 di-and tri-methylation detected by chromatin-immunoprecipitation(CHIP)on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions.Small RNAs were more abundant in the het erochromatic regions.To validate these findings,H3K4 trimethylation,H3K9 acetylation,H4K12 acetylation,and H3K9 di-and tri-methylation of 19 Individual genes were measured by Chlp-PCR.Genes in heterochromatic regions had elevated H3K9 di-and tri-methylation while genes in euchromatic regions had elevated levels of the other three modIfications.We also assayed cytosine methylation of these genes using the restriction enzymes McrBC,Hapll.and Msp I.This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated.These results suggest that IocaI transcriptional activity may retiect the organization of the corresponding part of the chromosome.They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptionaI activity.
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