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Reverse Chromatin Immunoprecipitation (R-ChIP) to investigate the upstream regulators of a specific gene（CAT3 Promoter)-880

ABSTRACT: This project aim to explore the gene-centered approaches to investigate the interaction between DNA and protein

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Whole Body

SUBMITTER: Xuejing Wen

LAB HEAD: Yucheng Wang

PROVIDER: PXD020755 | Pride | 2020-11-04

REPOSITORIES: Pride

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			Action	DRS
	P20200500880_1.dat	Other
	P20200500880_1.mgf	Mgf
	P20200500880_1.raw	Raw
	P20200500880_2.dat	Other
	P20200500880_2.mgf	Mgf

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Reverse Chromatin Immunoprecipitation (R-ChIP) to investigate the upstream regulators of a specific gene（CAT3 Promoter）-722

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Project description: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.

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Ca2+/S100 proteins act as upstream regulators of the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein).

Project description:The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca(2+)/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca(2+)/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca(2+)/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca(2+)/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca(2+)-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.

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Reverse phase protein arrays (RPPAs) (in vivo)

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