The Biological Function Of Zhangfei In Cellular ER Stress Response

The basic leucine zipper (bZIP) transcription factor Zhangfei (also called CREBZF or ZF) was identified through its interaction with Herpes Simplex Virus-1 (HSV-1) related cellular protein HCF-1. Researchers previously found that Zhangfei forms a heterodimer with ATF4 to play a role in the mammalian unfolded protein response. It is necessary to know about Zhangfei's regulation mechanism under UPR by focusing on the transcription, translation and protein function of Zhangfei. Here, a series of Zhangfei plasmids were construced, the real-time PCR, dual luciferase assay,western blotting and chromatin immunoprecipitation was used to research about the transcription and translation modulation of Zhangfei were conducted, the followed is the research method and results:1. Here report the identification of 213-base alternate mRNA splicing site at the 3′end of Zhangfei. The protein translated from the spliced mRNA will add an IFFFR tail to the C-terminus of the known Zhangfei isoforms. Also found the presence of a conserved in-frame ATG upstream of the currently known translation initiation site, which would add an additional 82-amino acid to the N-terminal. Therefore, Zhangfei can produce four isoforms, lZF, lZF-IFFFR, sZF and sZF-IFFFR.2. pGL3-Basic was used as a backbone and constructs a series of luciferase expression vector including different length fragement of Zhangfei promoter, MDCK cells were used for transfection.MDCK cells were transfected with a series of different Zhangfei promoter for 24 hours and treated with Tm or Tg for 24 hours, harvested protein and conducted dual luciferase assy, the results showed that -1767—-1112bp is essential for the basic transcription of Zhangfei promoter, however, these sequence can not response the stress induced by Tm or Tg. It was well known that nutrients, particularly amino acids, were involved in the control of gene expression. Here examined the molecular mechanisms involved in the regulation of Zhangfei expression upon amino acid deprivation using a transient expression assay, the results showed that transcriptional activation of the Zhangfei gene is regulated by amino acid deprivation, especially regulated by leucine deprivation. Furthermore, a cis-positive element, 5′-ATTCACTCA-3′,is essential for this response on the Zhangfei promoter. This Zhangfei specific sequence is the first described that can regulate a basal promoter in response to amino acid deprivation and therefore can be called an amino acid response element (AARE).3. Western blotting result showed that the constructing vector of pcFLAG-lZF, pcFLAG-lZF-IFFFR, pcFLAG-sZF and pcFLAG-sZF-IFFFR can express four isoforms of Zhangfei accordingly. Mutagenesis studies proved that lZF (SMILE-L) and lZF-IFFFR isoforms can produce the short sZF (SMILE-S) and sZF-IFFFR respectively by the alternate usage of translation initiation site. Also it was found that transcription levels of CREBZF were induced by ER stress. Interestingly, the induction of sZF-IFFFR isoform was identified by tunicamycin treatment and leucine deprivation, both of which are able to induce ER stress.4. CHOP, a gene involved in cell death, was identified as a direct target gene of lZF-IFFFR and sZF-IFFFR. sZF-IFFFR appeared to be a more potent activator of CHOP than lZF-IFFFR. Overexpression of both lZF-IFFFR and sZF-IFFFR activated transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, also called amino acid response element (AARE). shRNA knockdown of overexpressed sZF-IFFFR inhibited transcriptional activity of sZF-IFFFR on the CHOP promoter. Chromatin immunoprecipitation assays showed that sZF-IFFFR physically associated with the CHOP promoter. Overexpression of both lZF-IFFFR and sZF-IFFFR induced apoptosis by measuring caspase-3 activity, likely through activation of expression of CHOP, in both ER stress- and non-ER stress-induced apoptotic conditions.Overall, we report the identification of four CREBZF isoforms. We propose that two Zhangfei isoforms, lZF-IFFFR and sZF-IFFFR, are novel transcription factors that play roles in the CHOP-mediated apoptosis signaling pathway.