Repression of the c-myb gene by WT1 protein in T and B cell lines.

Stacey Mccann,Linda M Boxer,Janet Sullivan,Juan Guerra,Magdalena Arcinas

doi:10.1074/jbc.270.40.23785

Abstract

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.

Highlights

§ Supported by the Stanford University School of Medicine Medical Scholars Program and by a Howard Hughes research training fellowship for medical students
In the B cell line, DHL-9, this region did not show any protection, but a similar sequence extending from Ϫ455 to Ϫ446 was protected (Fig. 2)
Mutation of the EGR-1/WT1 site accounted for all of the negative regulatory activity in the region from Ϫ455 to Ϫ444 in DHL-9 cells. Both Sequences Bind the WT1 Protein—Because WT1 has been shown to act as a transcriptional repressor, we investigated whether it was responsible for the negative activity associated with the EGR-1/WT1 sites in the c-myb 5Ј-flanking region

Summary

Introduction

§ Supported by the Stanford University School of Medicine Medical Scholars Program and by a Howard Hughes research training fellowship for medical students. An important mechanism for regulation of mouse c-myb expression is a block to transcription elongation within the first intron of the c-myb locus, recognized as a pause site (9 –11). Recent studies conducted in mouse T cell lines suggest that murine c-myb expression is dependent on a GC-rich sequence of the 5Ј-flanking region and that the c-myb promoter is functional in diverse T cell lines [14]. We have shown that two Myb binding sites function as negative regulators of c-myb expression in T cell lines [15]. We show that WT1 binds to this site in vitro and, by cotransfection experiments, that WT1 negatively regulates c-myb expression. We found that WT1 bound to this site in vitro, and, in cotransfection experiments, it negatively regulated c-myb expression in B cells through this site

Methods

Results

Conclusion