A Highly Active Homeobox Gene Promoter Regulated by Ets and Sp1 Family Members in Normal Granulosa Cells and Diverse Tumor Cell Types

Abstract

One mechanism by which normal cells become converted to tumor cells involves the aberrant transcriptional activation of genes that are normally silent. We characterize a promoter that normally exhibits highly tissue-and stage-specific expression but displays ubiquitous expression when cells become immortalized or malignant, regardless of their lineage or tissue origin. This promoter normally drives the expression of the Pem homeobox gene in specific cell types in ovary and placenta but is aberrantly expressed in lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas. By deletion analysis we identified a region between nucleotides ؊80 and ؊104 that was absolutely critical for the expression from this distal Pem promoter (Pem Pd). Site-specific mutagenesis and transfection studies revealed that this region contains two consensus Ets sites and a single Sp1 site that were necessary for Pem Pd expression. Gel shift analysis showed that Ets and Sp1 family members bound to these sites. Transfection studies demonstrated that the Ets family members Elf1 and Gabp and the Sp1 family members Sp1 and Sp3 transactivated the Pem Pd. Surprisingly, we found that Sp3 was a more potent activator of the Pem Pd than was Sp1; this is unusual, because Sp3 is either a weak activator or a repressor of most other promoters. Activation by either Elf1 or Gabp required an intact Sp1 family member binding site, suggesting that Ets and Sp1 family members cooperate to activate Pem Pd transcription. Expression from the Pem Pd (either transiently transfected or endogenous) depended on the Ras pathway, which could explain both its Ets-and Sp1-dependent expression in normal cells and its aberrant expression in tumor cells, in which ras protooncogenes are frequently mutated. We suggest that the Pem Pd may be a useful model system to understand the molecular mechanism by which a tissue-specific promoter can be corrupted in tumor cells.

The Rhox genes

“…In the future, it will be important to determine whether other members of the large ETS family besides GABP control Pd transcription. The ETS family member ELF1 binds to the Pd, but its functional role in Pd transcription is not known (Rao et al 2002a). Interestingly, ELF1 upregulation has been associated with gynecological cancers of the cervix, uterus, and breast (Andrews et al 2008, Nicol et al 2008), but whether it has a role in ovarian tumors has not been determined.…”

“…The Pd is also responsible for expression of Rhox5 in the embryo proper and ES cells (Rao et al 2003;A Bhardwaj & MF Wilkinson 2009, unpublished observations). In contrast to its highly specific expression in normal tissues, the Pd is widely expressed in tumors, regardless of cell lineage or tissue of origin (Wilkinson et al 1990, Rao et al 2002a.…”

“…Subsequent work demonstrated that Rhox5 is not only expressed in a subset of T-cell lymphomas, but it is widely expressed in tumors derived from many different cell types and tissue origins (Wilkinson et al 1990, Maiti et al 1996b, Wayne et al 2002a. Its ubiquitous expression in tumors may derive, in part, from its ability to be induced by the Ras proto-oncogene (Rao et al 2002a, MacLean et al 2005b. RHOX5 may also have a causal role in tumor formation, as it induces an immune response in mouse tumors (Ono et al 2000), and it physically interacts with proteins involved in malignancy (Lemmens et al 2001, Guo et al 2005).…”

“…A key signal transduction event required for Pd transcripion is initiated by the G-protein RAS (Rao et al 2002a, MacLean et al 2005b). Gain-of-function evidence for this is that transient transfection of a constitutive form of RAS induces Pd expression in primary granulosa cells, mesenchymal stem cells, and tumor cells.…”

“…A minimal element required for Pd transcription has been defined (w100 nt upstream of its transcription start site) that is only 22 nt in length (Rao et al 2002a, MacLean et al 2005b). This 22-nt element is sufficient to activate the transcription of an inactive heterologous promoter in primary granulosa cells and tumor cells.…”