Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA).

Abstract

The octamer sequence ATGCAAAT is found in the promoters of immunoglobulin (Ig) heavy and light chain genes and in the heavy chain enhancer and is a major determinant of the cell type specific expression of Ig genes in B cells. An apparent paradox is that the same sequence serves as an upstream promoter or enhancer element in a variety of housekeeping genes such as the histone H2B and U snRNA genes. The differential usage of this regulatory sequence motif is thought to be mediated by different species of octamer binding proteins. One species of 100 kd, designated OTF-1, is present in all cell types and may exert its activating function only when it can interact with additional adjacent transcription factors. The lymphoid cell specific octamer binding protein of 60 kd (OTF-2A) specifically stimulates Ig promoters which consist essentially of a TATA-box and an octamer sequence upstream of it. Here we present evidence for yet another B cell specific octamer binding protein of 75 kd (OTF-2B). From several findings, including the absence of OTF-2B (but not OTF-2A) from a lymphocyte line that cannot respond to the IgH enhancer, we propose a role of the novel octamer factor in the long range activation by the IgH enhancer. We have used the proteolytic clipping bandshift assay (PCBA) technique to distinguish the three different forms found in B cells. This analysis indicates that the 75 kd-species OTF-2B is closely related to the 60 kd species OTF-2A.