Generation of Med28 Specific Monoclonal Antibodies

Yu, Min; Cho, Jin Gu; Kim, Kwang Il; Jo, Yeong Joon; Sung, Jong Hwan; Yang, Hyun-Kwon; Park, Sang Gyu

doi:10.1089/mab.2014.0068

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…Specimens were ductal carcinoma between stage 1 and 2. Immunohistochemical staining was performed as described previously [ 48 ]. Sections (5 μm) were stained for 1 h at RT with anti-p53 and anti-ZNF224 antibodies diluted with 1/50.…”

Section: Methodsmentioning

confidence: 99%

ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

et al. 2016

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ZNF224 is a Krüppel-associated box-containing zinc-finger protein which represses gene transcription by interacting with various co-repressors. However, its consensus DNA sequences and target genes are not fully identified. In this study, we identified and characterized consensus DNA sequences containing 5′-CAGC-3′; recognized by ZNF224 through ChIP-sequencing, which further confirmed by ELISA, SPR, qPCR, and luciferase activity assay. ZNF224 increased miR-663a transcription by binding to miR-663a promoter, which in turn binds to 3′; UTR of p53 and p21 to decrease their expression. miR-663a antagonist abolished ZNF224-mediated suppression of p21 and p53, resulting in the enhanced apoptosis by CPT. The analyses using human breast ductal carcinoma tissues exhibited that the expression of ZNF224 and miR-663a was increased in cancer compared to non-cancer region. Consequently, ZNF224 increases cell survival and decreases apoptosis by decreasing the expression of p53 and p21 via miR-663a as a transcriptional activator. Taken together, we identified and characterized DNA binding element of ZNF224, and its target genes, miR-663a, which provides a novel insight in the down-regulation of p21 and p53 via miR-663a by ZNF224 in breast cancer.

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Section: Methodsmentioning

confidence: 99%

ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

et al. 2016

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“…However, cancer genomics data, where MED28 was found to be amplified in various cancers, including breast cancer, ovarian cancer, pancreatic cancer, sarcoma, lung adenoma, prostate cancer, and melanoma, clearly indicate that the overexpression of MED28 is closely related to cancer progression (Supplementary Figure S4A). In addition, the analyses of RNA-seq data from various cancers suggest that the gene amplification of MED28 closely correlates with an increase in MED28 mRNA expression (Supplementary Figure S4B) [10,36,37].…”

Section: Discussionmentioning

confidence: 99%

MED28 Over-Expression Shortens the Cell Cycle and Induces Genomic Instability

Cho

Choi

Lee

et al. 2019

IJMS

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The mammalian mediator complex subunit 28 (MED28) is overexpressed in a variety of cancers and it regulates cell migration/invasion and epithelial-mesenchymal transition. However, transcription factors that increase MED28 expression have not yet been identified. In this study, we performed a luciferase reporter assay to identify and characterize the prospective transcription factors, namely E2F transcription factor 1, nuclear respiratory factor 1, E-26 transforming sequence 1, and CCAAT/enhancer-binding protein β, which increased MED28 expression. In addition, the release from the arrest at the G1−S or G2−M phase transition after cell cycle synchronization using thymidine or nocodazole, respectively, showed enhanced MED28 expression at the G1−S transition and mitosis. Furthermore, the overexpression of MED28 significantly decreased the duration of interphase and mitosis. Conversely, a knockdown of MED28 using si-RNA increased the duration of interphase and mitosis. Of note, the overexpression of MED28 significantly increased micronucleus and nuclear budding in HeLa cells. In addition, flow cytometry and fluorescence microscopy analyses showed that the overexpression of MED28 significantly increased aneuploid cells. Taken together, these results suggest that MED28 expression is increased by oncogenic transcription factors and its overexpression disturbs the cell cycle, which results in genomic instability and aneuploidy.

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“…Constructs expressing Flag-ZNF224 (12), Myc-MED28 (18), and 6x His-MED28 (18) were previously verified. HA-tagged N-terminus of ZNF224 construct was kindly provided by Dr. Constanzo (University of Naples Federico II, Naples, Italy).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, it was shown that ZNF224 downregulates the transcription of p53 and p21, and that MED28 is localized to the cytosol and nucleus (12,18). In addition, it is known that ZNF224 primarily localizes to the nucleus and forms a punctate pattern (12).…”

Section: Nucleus-specific Interactions Of Znf224 With Med28mentioning

confidence: 99%

MED28 increases the colony‑forming ability of breast cancer cells by stabilizing the ZNF224 protein upon DNA damage

2017

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Abstract. The regulation of gene expression by transcription factors serves a critical function in cell proliferation. Zinc-finger protein 224 (ZNF224), a Krüppel-associated-box-containing zinc finger protein, is known to serve a crucial function in integrating the transcriptional co-factors that activate transcriptional regulation pathways in the cell. A previous study demonstrated that ZNF224 enhances cell proliferation by downregulating the expression of p21 and p53. The present study identified mediator complex subunit 28 (MED28) as a potential binding partner for ZNF224; this was confirmed by co-immunoprecipitation and a surface plasmon resonance assay. Additionally, the KRAB domain at the N-terminal of ZNF224 interacts with the MED domain of MED28. Bimolecular fluorescence complementation analysis revealed that ZNF224 associates with MED28 in the nucleus. In addition, ZNF224 was rapidly degraded upon treatment with the DNA-damaging agent camptothecin (CPT). Transient overexpression of MED28 inhibited the CPT-mediated degradation of ZNF224, resulting in increased colony formation by MCF-7 cells. The molecular mechanisms that underlie the biological outcomes of MED28 expression have not yet been fully elucidated. The present study provides molecular evidence for the function of ZNF224 and MED28 in the DNA-damage response.

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Generation of Med28 Specific Monoclonal Antibodies

Cited by 3 publications

References 12 publications

ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

MED28 Over-Expression Shortens the Cell Cycle and Induces Genomic Instability

MED28 increases the colony‑forming ability of breast cancer cells by stabilizing the ZNF224 protein upon DNA damage

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