Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

Takahiro Kawanabe,Etsuko Itabashi,Keiichi Okazaki,Elizabeth S Dennis,Kenji Osabe,Ryo Fujimoto

doi:10.1266/ggs.15-00058

Abstract

Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.

Highlights

Epigenetic regulation is defined as changes in gene activity that are inherited through cell divisions without alteration of the DNA sequence
H3K9me2 is observed in pericentromeric heterochromatin regions, especially in transposable elements (TEs), and H3K27me3 is observed in genic regions with a low level of transcription and in a tissuespecific manner (Turck et al, 2007; Zhang et al, 2007; Zhang et al, 2008)
To identify regions having H3K36me3, we determined the sequences of 32 DNA fragments having H3K36me3, and approximately 50% of the fragments (17/32 genes) were in genic regions

Summary

Introduction

Epigenetic regulation is defined as changes in gene activity that are inherited through cell divisions without alteration of the DNA sequence. Alteration of chromatin structure, which causes changes in transcription, is regulated by various post-translational modifications of Genome-wide profiles of epigenetic information define the epigenome, and are determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq), especially to detect methylation and acetylation of lysine residues on histone H3 because histone H3 undergoes the most extensive modification. In Arabidopsis thaliana, the genome-wide distribution patterns of histone modifications such as H3K4me (methylation of the 4th lysine of H3), H3K9me, H3K27me and H3K36me have been examined (Turck et al, 2007; Zhang et al, 2007, 2009; Bernatavichute et al, 2008; Oh et al, 2008; Roudier et al, 2011). H3K4me and H3K36me are associated with gene activation, while H3K9me and H3K27me are associated with gene repression, in A. thaliana

Objectives

Methods

Results

Conclusion