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

Manjeet K Rao,Sourindra Maiti,Honnavara N Ananthaswamy,Miles F Wilkinson

doi:10.1074/jbc.m203374200

Manjeet K Rao, Sourindra Maiti + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m203374200

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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.

Highlights

Encodes a large group of transcription factors that each contain a 60-amino acid DNA-binding motif termed a homeodomain
Our data suggest that the Pem Pd is normally a tissue-specific promoter that is aberrantly expressed in diverse tumor cell types because it is activated by ubiquitously expressed transcription factors that are regulated in normal cells but constitutively activated in tumor cells
Diverse Tumor Cells Express Pem Transcripts from the Pem Pd—We showed previously [5] that despite its cell type-specific expression in normal cells, Pem is ubiquitously expressed in a variety of tumors cell lines regardless of their lineage

Summary

Introduction

Encodes a large group of transcription factors that each contain a 60-amino acid DNA-binding motif termed a homeodomain. To determine the specificity of this Pem Pd promoter element, we transfected the minimal Ϫ112 Pem construct containing this element into the immature T cell lymphoma cell clones SL12.1 and SL12.3, both of which express little or no Pem transcripts from the endogenous Pem gene (Fig. 3A).

Results

Conclusion