Studies have shown that the transformation of benign prostate epithelial cells into adenocarcinoma is consistently associated with loss of the ability to accumulate intracellular zinc through down-regulation of the hZip1 zinc transporter (Franklin et al., 2003
; Rishi et al., 2003
; Franklin et al., 2005
). To further understand this process, we explored the mechanisms regulating hZip1 gene expression.
hZip1 is a housekeeping gene essential to normal cell function and appears to be evolutionarily conserved (Dufner-Beattie et al., 2003
). With the conservation of the protein and nucleotide structure, there also may be common mechanisms of transcriptional regulation across species. Indeed, our comparison of human and mouse Zip1 gene 5′-flanking region sequences revealed a high degree of similarity, especially near the transcription initiation sites.
To locate the core promoter in PC-3 human prostate cancer cells, we obtained an 830-bp DNA fragment representing the predicted promoter region and first exon of the hZip1 gene. The minimal hZip1 core promoter region was identified through luciferase assays as a −224/+82 fragment relative to the transcription start site. This fragment is responsible for basal hZip1 promoter activity, which is comparable to that of the SV40 promoter. To our knowledge, the core promoter region has not been previously identified in the literature.
The experimentally determined by 5′RACE main hZip1 transcription initiation site was −836; whereas the initiation site identified for the AK075257 transcript in the NCBI database is −858. The hZip1 gene appears to lack canonical CCAAT, TATA box, and initiator elements in its GC-rich promoter region. Such promoters containing CpG islands are known to exhibit multiple transcription initiation sites that can be alternatively active (Swick et al., 1989
; Geng and Johnson, 1993
; Liu et al., 2006
; Sandelin et al., 2007
). In the case of PC-3 prostate cancer cells, our findings suggest an alternative main transcription initiation site for hZip1. Transcription continued when the main initiation site was inactivated. We found that transcription of a hZip1(−224/−30) fragment lacking the main transcription initiation site remained 30% active compared to baseline. The continued transcription of a housekeeping gene despite disruption of its main initiation site is highly important for cell survival.
Our computer analysis, functional luciferase reporter assays, mutations analysis, GelSupershift, and ChIP experiments identified SP1 as an important transcription factor in the control of hZip1 expression, particularly at the −8 and −80 positions. Previous in vitro studies have shown that SP1 can direct transcription initiation from heterogeneous TSSs in core promoters that lack both TATA and initiator elements (Smale and Kadonaga, 2003
; Muckenfuss et al., 2007
; Yang et al., 2007
). Moreover, GC-rich promoters are characterized by multiple SP1 binding sites, and their activity is often controlled by SP1 (Kingsley and Winoto, 1992
; Toonen et al., 1996
; Seyed and Dimario, 2007
; Yu et al., 2007
). The SP1 family of proteins has been implicated in a host of essential biological processes, including apoptosis, cell growth inhibition, differentiation, and carcinogenesis (Lee et al., 2005
Gel Supershift, luciferase, and ChIP assays also identified CREB1 as an important regulator of hZip1 promoter activity. Signaling pathways through CREB proteins are known to be important for cell survival and proliferation (Mayr and Montminy, 2001
). In general, CREB binding sites are located between the −50 and −150 positions relative to a start site and can produce both basal and inducible transcription (Quinn, 1993
; Felinski and Quinn, 1999
; Mayr and Montminy, 2001
Furthermore, our data suggest an additive transcriptional regulation of the hZip1 promoter by SP1 and CREB1 transcription factors. Concomitant SP1 and CREB binding site mutation led to an 80% loss of hZip1 promoter transcriptional activity; whereas isolated mutations of SP1 or CREB binding sites resulted in loss of approximately 75% and 50% of activity, respectively.
The data suggesting that SP1 and CREB1 additively regulate hZip1 promoter activity was supported by ChIP experiments, which demonstrated that these transcription factors specifically bind with the hZip1 core promoter −174/+51 region.
Additionally, we have shown that specific knockdown of SP1 and CREB1 proteins using siRNA duplexes leads to impairment of hZip1 gene transcription.
To avoid the anti-tumor effects of intracellular zinc, malignant prostate cells silence hZip1 gene expression (Rishi et al., 2003
; Franklin et al., 2005
). Re-establishment of normal intracellular zinc levels is an attractive target for prostate cancer therapy. This study begins to identify the mechanisms responsible for constitutive expression of the hZip1 gene. Increased understanding of the regulatory mechanisms behind hZip1 gene expression could provide the basis for further research to produce therapeutic up-regulation of hZip1 expression in prostate adenocarcinoma.