Abstract
DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. In the present study, we developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are then identified using mass spectrometry. Using R-ChIP, we identified 439 proteins that potentially bind to the promoter of the Arabidopsis thaliana gene AtCAT3 (AT1G20620). According to functional annotation, we randomly selected 5 transcription factors from these candidates, including bZIP1664, TEM1, bHLH106, BTF3, and HAT1, to verify whether they in fact bind to the AtCAT3 promoter. The binding of these 5 transcription factors was confirmed using chromatin immunoprecipitation quantitative real-time PCR and electrophoretic mobility shift assays. In addition, we improved the R-ChIP method using plants in which the DNA of interest had been transiently introduced, which does not require the T-DNA integration, and showed that this substantially improved the protein capture efficiency. These results together demonstrate that R-ChIP has a wide application to characterize chromatin composition and isolate upstream regulators of a specific gene.
Highlights
DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging
The first step is crosslinking of proteins and chromatin using formaldehyde; the second step is isolation of nucleus; the third step is shearing the chromatin within 1 kb in length by sonication
The results showed that the truncated promoter of AtCAT3 was substantially enriched for all five Transcription factors (TFs), suggesting that the AtCAT3 promoter was bound by bZIP1664, TEM1, bHLH106, BTF3, and HAT1 in Arabidopsis plants (Fig. 7b–f)
Summary
DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. We developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP) This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are identified using mass spectrometry. Until now, limited genecentered in vivo methods have been reported, including proteomics of isolated chromatin segments (PICh)[8], chromatin isolation by RNA purification (ChIRP)[9], protein–RNA interaction mapping assay (PRIMA)[10], and Y1H Among these technologies, ChIRP and PRIMA are used to identify proteins that bind to RNA. We developed a method to capture proteins from specific formaldehyde cross-linked chromatin or DNA regions and to identify these proteins using mass spectrometric analysis (MS) Using this method, the proteins binding to the promoter region of the Arabidopsis thaliana gene AtCAT3. We called this technology Reverse Chromatin Immunoprecipitation (R-ChIP), because it is the opposite of ChIP, using the DNA to retrieve the proteomic information
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