Mass spectrometry-based proteomic capture of proteins bound to the MACC1 promoter in colon cancer
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RESEARCH PAPER
Mass spectrometry‑based proteomic capture of proteins bound to the MACC1 promoter in colon cancer Yahui Huang1,2,4 · Yi Xiang2 · Zhongpeng Xie1,2,5 · Yuxiang Cai1,2 · Qiongzhi Yang1,2 · Huichao Huang3 · Zhuchu Chen3 · Zhefeng Xiao3 · Qiongqiong He1,2 Received: 16 January 2020 / Accepted: 7 June 2020 © Springer Nature B.V. 2020
Abstract MACC1 (metastasis associated in colon cancer 1) is a key driver that induces metastasis in colon cancer. However, the mechanisms by which MACC1 expression is transcriptionally regulated and the factors enriched at the MACC1 promoter remain largely unknown. The binding of proteins to specific DNA sites in the genome is a major determinant of genomic maintenance and the regulation of specific genes. The study herein utilized two methods to study the binding proteins of the MACC1 promoter region in colon cancer. Specifically, we adopted CRISPR-based chromatin affinity purification with mass spectrometry (CRISPR-ChAP-MS) and a biotin-streptavidin pulldown assay coupled with MS to identify the specific proteome bound to the MACC1 promoter in two colon cell lines with different metastatic potential. A total of 24 proteins were identified by CRISPR-ChAP-MS as binding to the MACC1 promoter, among which c-JUN was validated by ChIP-PCR. A total of 739 binding protein candidates were identified by biotin-streptavidin pulldown assays coupled with MS, of which HNF4G and PAX6 were validated and compared for their binding to the same promoter sites in the two cell lines. Our studies suggest distinctive proteomic factors associated with the MACC1 promoter in colon cells with different metastatic potential. The dynamic regulatory factors accumulated at the promoter of MACC1 may provide novel insights into the regulatory mechanisms of MACC1 transcription. Keywords Colon cancer · MACC1 · CRISPR-ChAP-MS · Biotin-streptavidin pulldown · Proteomics
Introduction Gene expression is regulated via interactions between regulatory proteins and genomic elements. Transcriptional regulation not only refers to histone posttranslational Yahui Huang and Yi Xiang have contributed equally to this work and should be considered co-first authors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10585-020-10045-z) contains supplementary material, which is available to authorized users. * Zhefeng Xiao [email protected] * Qiongqiong He [email protected] 1
Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China
School of Basic Medical Sciences, Central South University, Changsha, Hunan, People’s Republic of China
2
modifications (PTMs) at a specific chromosomal location but also is a function of the transcriptional factors binding to the region. The composition of a specific “local epiproteome” in the regulatory region of a gene affects whether a gene is actively transcribed, repressed, or held in an intermediary state [1]. Identifying the complete inventory of a specific epiproteome, a
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