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

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: PXD020772 | Pride | 2020-11-04

REPOSITORIES: Pride

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			Action	DRS
	P20200500722_4.dat	Other
	P20200500722_4.mgf	Mgf
	P20200500722_4.raw	Raw
	P20200500722_5.dat	Other
	P20200500722_5.mgf	Mgf

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

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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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Identification and characterization of DNA sequences that restrict genomic binding of the glucocorticoid receptor

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Reverse phase protein arrays (RPPAs) (in vitro)

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Nucleolar Stress Functions Upstream to Stimulate Expression of Autophagy Regulators.

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Identification of key genes and upstream regulators in ischemic stroke.

Project description:IntroductionIschemic stroke (IS) causes severe neurological impairments and physical disabilities and has a high economic burden. Our study aims to identify the key genes and upstream regulators in IS by integrated microarray analysis.MethodsAn integrated analysis of microarray studies of IS was performed to identify the differentially expressed genes (DEGs) in IS compared to normal control. Based on these DEGs, we performed the functional annotation and transcriptional regulatory network constructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the expression of DEGs.ResultsFrom two Gene Expression Omnibus datasets obtained, we obtained 1526 DEGs (534 up-regulated and 992 down-regulated genes) between IS and normal control. The results of functional annotation showed that Oxidative phosphorylation and Alzheimer's disease were significantly enriched pathways in IS. Top four transcription factors (TFs) with the most downstream genes including PAX4, POU2F1, ELK1, and NKX2-5. The expression of six genes (ID3, ICAM2, DCTPP1, ANTXR2, DUSP1, and RGS2) was detected by qRT-PCR. Except for DUSP1 and RGS2, the other four genes in qRT-PCR played the same pattern with that in our integrated analysis.ConclusionsThe dysregulation of these six genes may involve with the process of ischemic stroke (IS). Four TFs (PAX4, POU2F1, ELK1 and NKX2-5) were concluded to play a role in IS. Our finding provided clues for exploring mechanism and developing novel diagnostic and therapeutic strategies for IS.

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