Rabbit Anti-Human ZNF746 (a.a. 186-531) Polyclonal antibody

Background ZNF746 and ZNF777 belong to a subset of the large Kruppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the "domain of unknown function 3669" (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson's disease. Results Here we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669's impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved "MLE" sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein. Conclusion DUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

Although ZNF746, also known as Parkin-interacting substrate (PARIS), has been reported to suppress peroxisome proliferator-activated receptor coactivator-1 (PGC-1) and its target gene NRF-1 leading to the neurodegeneration in Parkinson's disease, its function in tumorigenesis has yet to be investigated. Thus, in the present study, the role of ZNF746 in the invasion and epithelial to mesenchymal transition (EMT) in H460 non-small cell lung cancer (NSCLC) cells was investigated. Invasion assay showed that inhibition of ZNF746 using siRNA transfection inhibited the invasion of H460 NSCLC cells using Boyden chamber. Quantitative PCR (qPCR) analysis revealed that the silencing of ZNF746 attenuated the expression of matrix metalloproteinase (MMP)1, MMP2 and MMP9, but not MMP7, in H460 NSCLC cells. Immunoblotting assay revealed that the expression of E-cadherin and -catenin of epithelial phenotype was upregulated, while Slug was downregulated in ZNF746 siRNA-transfected H460 NSCLC cells. Accordingly, the mRNA expression of E-cadherin was upregulated while vimentin or Slug, Twist, ZEB as EMT key transcriptional factors were suppressed in ZNF746 siRNA-transfected H460 NSCLC cells. Also, mRNA expression of transcriptional marker Nanog and Octamer-binding transcription factor 4 (OCT4), known to enhance malignancy and metastasis in lung adenocarcinoma, was suppressed in ZNF746 siRNA-transfected H460 NSCLC cells. Notably, the endogenous expression of ZNF746 was induced in parallel with Twist at the protein level during hypoxia. Overall, our findings suggest that inhibition of ZNF746 suppresses the invasion and EMT molecules in H460 NSCLC cells and ZNF746 may be an important target molecule in lung tumorigenesis.