Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications

<div><p>Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including <i>Drosophila melanogaster</i>. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., <i>Bactrocera dorsalis</i>, <i>Ceratitis capitata</i>, <i>Zeugodacus cucurbitae</i> and <i>Bactrocera tryoni</i>. We also report an example ChIP-seq dataset for <i>B</i>. <i>tryoni</i>, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.</p></div

Summary sequence data from a single Illumina run (several runs were performed for different antibodies).

<p>Summary sequence data from a single Illumina run (several runs were performed for different antibodies).</p

Schematic representation of ChIP workflow and crucial steps for an effective crosslinking and immunoprecipitation.

<p>Schematic representation of ChIP workflow and crucial steps for an effective crosslinking and immunoprecipitation.</p

Buffers used for cell lysis and immunoprecipitation for ChIP sequencing of tephritid fruit flies.

<p>Buffers used for cell lysis and immunoprecipitation for ChIP sequencing of tephritid fruit flies.</p

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications - Fig 2

<p>(A) An example of product size after sonication using Bioruptor at 30 sec on, 30 sec off at high speed for 70–80 minutes; (B) Conjugation of (a) H3K27me3 (b) H3K27 acetylation (c) mock in <i>B</i>. <i>tryoni</i> checked for <i>Obp 99c</i> and <i>Unigene266</i> genes; (C) Bioanalyzer image showing the size and concentration of chromatin DNA immunoprecipitated with H3K27me3 (D) Chromatin DNA immunoprecipitated with H3K27me3 after enrichment.</p

An example of fruit fly output DNA recovered after conjugation of specific antibodies when using known concentrations of cross-linked DNA.

<p>An example of fruit fly output DNA recovered after conjugation of specific antibodies when using known concentrations of cross-linked DNA.</p

Output DNA of 200–300 bp size (enriched product) obtained from a starting material of 10 ng of antibody conjugated DNA from <i>Bactrocera tryoni</i> head tissues using Illumina Truseq ChIP sample preparation kit.

<p>Output DNA of 200–300 bp size (enriched product) obtained from a starting material of 10 ng of antibody conjugated DNA from <i>Bactrocera tryoni</i> head tissues using Illumina Truseq ChIP sample preparation kit.</p

An example of genomic regions displaying enrichment for H3K4me3, H3K27Ac, H3K36me1 and H3K36me3 histone proteins in <i>Bactrocera tryoni</i> when visualised using the ‘Sushi.R’ R/Bioconductor package (SPMR—Sequences Per Million Reads).

<p>‘Input’ samples that were not conjugated with any antibodies served as ‘control’ to compare histone modifications in antibody conjugated samples. Differential peak height and width represents enrichment of histone proteins at various genomic regions.</p

List of antibodies used for protocol optimization and their product codes for chromatin immunoprecipitation sequencing of tephritid fruit flies.

<p>List of antibodies used for protocol optimization and their product codes for chromatin immunoprecipitation sequencing of tephritid fruit flies.</p

Schematic of pBIN19-mGFP-ER and Tn5393 and their integration site in 16c.

<p><b>(A–B)</b> Schematic of genes within the T-DNA and transposon integrated in 16c genome. The sequence junctions between <i>N</i>. <i>benthamiana</i> genome-T-DNA-transposon-<i>N</i>. <i>benthamiana</i> genome are highlighted, GenBank accession number KY464890. <b>(C)</b> Log scale density plot of sequence reads aligning to T-DNA, transposon and flanking <i>N</i>. <i>benthamiana</i> genomic regions. <b>(D)</b> Location of primers used for amplification of various regions to confirm site of integration. <b>(E)</b> Log scale density plot of RNAseq reads aligning to T-DNA and transposon. <b>(F)</b> Gel image showing the endpoint (35 cycles) PCR amplicons of the T-DNA insert in 16c with LAB <i>N</i>. <i>benthamiana</i> used as a control. Expected sizes with primer pair B+E is 4174 nt and D+F is 4167 nt. NTC, no template control, 1 kb DNA Ladder (GeneRuler^™). <b>(G)</b> Leaves of <i>N</i>. <i>benthamiana</i> 16c photographed under UV light, showing mobile silencing, 14 days after local induction of RNAi at a lower leaf. Abbreviations are: RB: Right Border, pNOS: nopaline synthase promoter, <i>NPTII</i>: neomycin phosphotransferase II encoding gene, tNOS: nopaline synthase terminator, p35S: Cauliflower mosaic virus 35S promoter. IR: inverted terminal repeat, <i>tnpA</i> transposase gene, res: recombination region, <i>tnpR</i> resolvase gene, IS1133: an insertion element, <i>strA</i> and <i>strB</i>: streptomycin resistance genes, Nb Genome: Flanking DNA of <i>N</i>. <i>benthamiana</i>.</p