PJA2 Suppresses Colorectal Cancer Progression by Controlling HDAC2 Degradation and Stability
Zhihao ChenChaofan PengChi JinYe WangTuo WangPeng YangWen PengQingyang SunHengjie XuHongxu NieXiaowei WangJunwei TangYueming SunYifei Feng
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PJA2 is documented to degrade various substrates. Nevertheless, the role of PJA2 as an E3 ubiquitin-protein ligase in colorectal cancer (CRC) progression remains unexplored. The correlation between PJA2 mRNA levels and clinical characteristics is investigated using data from The Cancer Genome Atlas (TCGA) database. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) are utilized to evaluate PJA2 expression levels in CRC tissues. The biological functions of PJA2 are confirmed through colony formation assays and azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model of CRC, among other experimental approaches. The underlying molecular mechanisms of PJA2 action are elucidated using RNA sequencing (RNA-seq), co-immunoprecipitation (co-IP), proximity ligation assay (PLA), and chromatin immunoprecipitation (ChIP). Our research discovered that PJA2 is downregulated in CRC tissues and decreased PJA2 expression correlates with poor prognosis. Functionally, in vivo and in vitro experiments uncovered that PJA2 inhibits tumor cell proliferation and promotes apoptosis. Mechanistically, PJA2 recognized histone deacetylase 2 (HDAC2) via its RING-B-box domain (RBD) and bind to the N-terminal of HDAC2, facilitating ubiquitination at the lysine 90 (K90) residue. PJA2-mediated degradation of HDAC2 counteracts the transcriptional repression of the interferon-induced protein with the tetratricopeptide repeats (IFIT) family, thereby suppressing CRC progression. The data demonstrates that PJA2 suppresses CRC progression through the PJA2/HDAC2/IFIT axis, and its expression is regulated by HDAC2, thus constituting a positive feedback loop. Consequently, PJA2 may serve as a potential therapeutic target for CRC, and interrupting this feedback loop can represent a viable treatment strategy to restrain CRC progression.Keywords:
Chromatin immunoprecipitation
Immunoprecipitation
Histone deacetylase 2
Chromatin immunoprecipitation (ChIP) experiments with differentiated adipocytes are challenging because lipid droplets interfere with immunoprecipitation efficiency. Here, the author describes optimized procedures to minimize the burden of lipid droplets by using hypotonic buffer to enrich nuclear fraction before formaldehyde crosslinking, thus increasing the sensitivity and specificity of ChIP experiments with differentiated adipocytes. The author also describes steps after fixation, including sonication, immunoprecipitation, washing, reverse-crosslinking, and purification. This protocol is compatible with ChIP-qPCR and ChIP-seq of various transcription factors and histone modifications. For complete details on the use and execution of this protocol, please refer to Hiraike et al. (2022).1.
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Abstract Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein‐DNA complexes are cross‐linked with formaldehyde ( UNIT ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein‐DNA cross‐links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co‐occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co‐occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae . Cells from a wide variety of organisms can be used.
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Abstract In the past few decades, numerous approaches have been developed to investigate protein‐protein and protein‐nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co‐immunoprecipitation (co‐IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two‐step co‐immunoprecipitation (TIP) technique. As compared to standard co‐IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein‐protein complex from Burkitt lymphoma cells and from primary human CD4 + T cells. In addition, this unit describes an application of TIP for the isolation of transcription‐factor‐bound chromatin. © 2018 by John Wiley & Sons, Inc.
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This protocol can be ued for chromatin immunoprecipitation of RNAPII and associated factors, as well as histones. The settings are given for HeLa cells and should be adapted for other cell types.
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To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.
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Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.
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DNA-protein interactions are a major challenge in the research of gene transcription regulation.Chromatin immunoprecipitation assay(CHIP)provides a powerful tool to analyze this interaction in vivo.This article summarizes the methods of ChIP assay,and highlights the application and recent progress in the application of this technique.
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Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including Drosophila . This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of Drosophila nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters.
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