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An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.

ABSTRACT: Sequential chromatin immunoprecipitation (ChIP) is commonly used to investigate DNA-protein and protein-protein interactions to a specific genomic region. However, it can be tricky to achieve a robust and reproducible signal with sequential ChIP. Here, we provide an optimized two-step ChIP protocol to quantify the in vivo associates of multiple proteins with the same DNA regulatory element. For complete details on the use and execution of this protocol, please refer to He et al. (2020).

SUBMITTER: He L

PROVIDER: S-EPMC8100613 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.

He Lingli L Yu Wentao W Zhang Wenxiang W Zhang Lei L

STAR protocols 20210427 2

Sequential chromatin immunoprecipitation (ChIP) is commonly used to investigate DNA-protein and protein-protein interactions to a specific genomic region. However, it can be tricky to achieve a robust and reproducible signal with sequential ChIP. Here, we provide an optimized two-step ChIP protocol to quantify the <i>in vivo</i> associates of multiple proteins with the same DNA regulatory element. For complete details on the use and execution of this protocol, please refer to He et al. (2020). ...[more]

PMID: 33997818

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Project description:BackgroundDuring ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening.ResultsUsing immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression.ConclusionsThe presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own promoter suggests the presence of autoregulation for RIN expression. ChIP-based analyses identified a novel RIN-binding CArG-box site that harbors a gene associated with RIN expression in its flanking region. These findings clarify the crucial role of RIN in the transcriptional regulation of ripening initiation and progression.

Dataset Information

An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.

Publications

An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.

Similar Datasets

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