Advancing Epigenetics Towards Systems Biology

Chromatin Immunoprecipitation Protocol to Analyze Histone Modifications in Arabidopsis thaliana (Prot 12)

Werner Aufsatz

Introduction

Eukaryotic chromatin is a complex of DNA and associated histone proteins which are involved in the higher order packaging of DNA into chromosomes. The chromatin state of a given DNA sequence influences transcriptional activity and replication timing and is regulated by potentially reversible covalent modifications of DNA and histones. Histone modifications at conserved lysine and arginine residues within the flexible N-terminal tails, such as phosphorylation, acetylation and methylation, specify a code which serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of crosslinked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation; ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes and is already routinely used in many labs. In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, I describe the method used in our laboratory to study histone modifications in the plant model organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers (Lawrence et al., 2004).

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Werner Aufsatz

Gregor Mendel Institute of Molecular Plant Biology - Austrian Academy of Sciences - Dr. Bohrgasse 3 - A-1030 Vienna, Austria

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Werner Aufsatz