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. 2012 Dec 7;8(1):48.
doi: 10.1186/1746-4811-8-48.

Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

Xin Lin  1 , Leïla Tirichine, Chris Bowler
Affiliations

Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

Xin Lin et al. Plant Methods. .

Abstract

In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.

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Figures

Figure 1
Figure 1
Outline of the ChIP QPCR protocol. Timing for each step is indicated between parentheses.
Figure 2
Figure 2
Sonication efficiency. Different times and number of cycles were used for sonication. After crosslinking, eluted DNA was loaded in a 2% gel to monitor the efficiency of sonication. From left to right, ladder, 10 times diluted sample containing 500000 cells/ml with 7 cycles 25 seconds ON and 1 minute OFF, original sample from 200 ml culture containing 5 million cells/ml, 4x and 2x diluted samples with 7 cycles, 25 seconds and 1 minute OFF and 4x diluted sample with 9 cycles, 30 seconds ON and 1 minute OFF.
Figure 3
Figure 3
Analysis of antibody specificity using peptide competition assays on western blots of P. tricornutum nuclear extracts. (A) Upper lane contains from left to right 1 μg of H3K4me2 antibody alone or with 0.25 μg of one of the different modified peptides, H3K4me1, H3K4me2 and H3K4me3. Lower lane contains H4 antibody as internal loading control. (B) Upper lane contains 1 μg of H3K9me2 antibody alone or with 0.25 μg of one of the different modified peptides, H3K9me1, H3K9me2 and H3K9me3. Lower lane contains H4 antibody as internal loading control.
Figure 4
Figure 4
H3K4me2 histone modification on different regions of genes and TEs in P. tricornutum (A) and T. pseudonana (B). % IP indicates the enrichment. H4 P: promoter region of H4 histone gene. H4 B: body region of H4 histone gene. PHY P: promoter region of Dph gene. PHY B: body region of Dph.
Figure 5
Figure 5
H3K9me2 histone modification on different regions of genes and TEs in P. tricornutum (A) and T. pseudonana (B). % IP indicates the enrichment. H4 P: promoter region of H4 histone gene. H4 B: body region of H4 histone gene. PHY P: promoter region of Dph gene. PHY B: body region of Dph. Mock values are indistinguishable from 0% in T. pseudonana.
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