The Function And Mechanism Of ZNF451in TGF-β Signaling Pathway

Members of the Transforming Growth Factor-β (TGF-β) family have diverse functions during cell proliferation, differentiation, apoptosis, embryonic development and organogenesis. Dysfunctions of TGF-β and related factors are linked to various human diseases. In cancer, TGF-β signaling plays an important role in suppressing cell proliferation through inhibition of cell cycle progression. TGF-β receptors and Smads are often inactivated in many types of cancer such as pancreatic and colorectal cancers. Parodoxically, TGF-β promotes cancer progression and metastasis during advanced stages of cancer.TGF-β signals are transduced by Smad proteins, which form the canonical TGF-β pathway. There are eight distinct Smad proteins, which are divided into three classes: the receptor-regulated Smads (R-Smads), the Co-mediator Smad (Co-Smad), and the inhibitory Smads (I-Smads). Smad4is the only Co-Smad in mammals. Upon phosphorylation by the TGF-β receptors, R-Smads form a complex with Smad4and are then translocated into the nucleus. In the nucleus, the activated Smad complex cooperates with diverse groups of DNA-binding factors and transcriptional cofactors to control gene transcription in a context-dependent manner.ZNF451(Zinc Finger Protein451) is a new member of zinc finger protein family. Classical zinc finger proteins contain the C2H2zinc finger motif that requires coordination of zinc ions to stabilize its structure. ZNF451contains11Kruppel-like C2H2-type zinc fingers. The N-terminus of ZNF451is very rich in acidic residues and contains a SUMO-interaction motif (SIM), and the C-terminus contains a ubiquitin-interacting motif (UIM). ZNF451is found in the PML (promyelocytic leukaemia) body, and regulates transcription through the SUMO-modification machinery. Depletion of ZNF451in prostate cancer cells significantly decreases expression of androgen receptor (AR) target genes.In this study, we determined how ZNF451participates in TGF-β signaling pathway. We first identified ZNF451as a novel Smad4-binding partner in a yeast two-hybrid screening. This interaction between ZNF451and Smad4was further validated in vivo and in vitro. Then, we took both gain-of-function and loss-of-function approaches to elucidate the physiological role of ZNF451in TGF-β signaling. We found that overexpression of ZNF451repressed expression of the TGF-β target genes, whereas depletion of ZNF451upregulated the TGF-β target genes. Furthermore, mutations of the sumoylation site Lys706or SIM in ZNF451did not change its inhibitory function in TGF-β signaling suggesting that ZNF451negatively regulates TGF-β signaling in a SUMO-modification-independent manner. Mechanistically, ZNF451exhibited no effects on Smad activation, yet it blocked the recruitment of p300to the Smad complex, and caused reduction of histone H3K9acetylation on the promoters of TGF-P target genes such as p15Ink4b and p21cip1. As a result of ZNF451overexpression, TGF-β-induced p15Ink4b and p21Cip1expression and consequent cell growth arrest were compromised, indicating oncogenic potential of ZNF451. Since TGF-β also induces epithelial mesenchymal transitions (EMT) in epithelial cells, we examined whether and how ZNF451participates in TGF-β-induced EMT. We found that ZNF451downregulated epithelial markers and upregulated mesenchymal markers in mammary epithelial MCF10A cells and lung epithelial A549cells. Notably, ZNF451directly mediated E-cadherin repression and also promoted cell migration in these cells in a TGF-β-independent manner.In summary, ZNF451regulates cellular functions in a TGF-β-dependent and-independent pathway. On one hand, ZNF451acts as a novel transcriptional corepressor for Smads in regulating TGF-β growth inhibitory and transcriptional responses. On the other hand, ZNF451directly induces EMT independently of TGF-β. This study suggests that ZNF451may be a critical player in regulating cancer progression.