Maintenance of Zn homeostasis is essential for health and cellular function; however, the transcriptional control of Zn responsive genes is currently poorly characterised. A novel Zinc Responsive Transcriptional element (ZRE) within the promoter region of the SLC30A5 Zn transporter gene was identified and an in silico search for the ZRE identified 6 human paralogous COBW domain-containing (CBDW) genes as potentially responsive to Zn. Furthermore, comparative genomic analysis of prokaryotic CBWD homologues has revealed a potential role in metal homeostasis and trafficking(Reference Haas, Rodinov and Kropat1).
The abundance of the CBWD transcript in Caco-2 cells, normalised to GAPDH, was measured by RT-qPCR to be ~50% lower (P<0.001) in cells treated for 24 h with 100 μm Zn (0.48±0.06, n 6) compared with 3 μm Zn (1.00±0.09, n 6). Zn-regulated CBDW transcription and the role of the ZRE was investigated using plasmid constructs comprising the region −1078 to +98 of the CBDW3 gene, relative to the start of transcription, upstream of the β-galactosidase reporter gene in the vector pBlueTOPO (Invitrogen). Reporter gene expression was lower (P<0.001) at 100 μm Zn (0.73±0.04, n 23) compared with 3 μm Zn (1.00±0.04; n 24) in transfected Caco-2 cells treated with Zn for 24 h. The response of the reporter gene to Zn was attenuated (P<0.01) by mutating the ZRE to a random sequence (1.00±0.03 at 3 μm Zn; 0.90±0.02 at 100 μm zinc; n 12), consistent with the ZRE having a role in mediating the transcriptional response to Zn.
Human CBWD3 protein, incorporating a C-terminal FLAG tag, was expressed transiently from a plasmid construct comprising the full open reading frame of the human CBDW3 gene in the vector pCMV6Entry (Origene) in chinese hamster ovary cells treated for 24 h with 3 or 100 μm Zn. Western-blot analysis using an anti-FLAG antibody indicated greater abundance (P<0.05) of the recombinant protein resolved by SDS-PAGE at the expected molecular weight (~44 kDa) in cells cultured at the higher Zn concentration (1.00±0.12 at 3 μm Zn; 2.57±0.36 at 100 μm Zn; derived by densitometric quantification of band intensity, n 3–4). Preliminary observations indicated increased abundance at the lower Zn concentration of an anti-FLAG immunoreactive band of a lower molecular weight (~30 kDa), possibly indicative of Zn-dependent cleavage.
These observed effects of Zn availability on the expression of the CBDW genes and CBDW protein are consistent with the view that the human CBDW proteins play a role in Zn homeostasis. Further studies, including the identification of CBDW protein-binding partners, description of tissue and sub-cellular distribution and effects of Zn and other divalent metals on these measures, may help to elucidate this role.
Funded by BBSRC grant ref. BB/F019637/1.
