Skip to main content
. 2006 Nov 13;27(2):579–594. doi: 10.1128/MCB.01192-06

FIG. 1.

FIG. 1.

The Ebf1 gene is regulated by two promoters, the distal α and proximal β promoters. (A) Schematic overview of the distal Ebf1α and proximal Ebf1β promoters. Two alternative first exons, exon 1a and exon 1b, are indicated. The proximal Ebf1β promoter is located within the first intron of Ebf1α mRNA and 879 nucleotides upstream of the ATG-β, which is spliced out in Ebf1α mRNA. Ebf1α mRNA and Ebf1β mRNA can be distinguished by RT-PCR with primer pairs specific for exon 1a or exon 1b sequences. Indicated are the positions of the PCR amplicons and of the primer used for primer extensions and S1 nuclease protection experiments. (B) Primer extension experiments show multiple bands corresponding to potential 5′ ends of Ebf1β mRNA located 879 nucleotides (nt) upstream of the ATG-β. Potential transcriptional start sites are visible only in 70/Z3 pre-B cells and in spleen, not in Ebf1-negative EL4 T cells. (C) S1 nuclease protection experiments show a similar pattern of protected Ebf1β 5′ ends as in the primer extension experiments. nt, nucleotides. (D) Alignment of the human and mouse Ebf1β promoter transcriptional start sites. (E) Quantitative RT-PCR analysis of EL4 T cells and sorted lymphoid cells shows the upregulation of Ebf1 mRNA during early B-cell development. HSC (Lin− IL-7R+ c-kithigh Sca-1high), CLP (Lin− IL-7R+ c-kitlow Sca-1low), pro-B cell Fr. A (B220+ AA4.1+ c-kit+ CD19−) and Fr. B and Fr. C according to the work of Hardy and Hayakawa (18) (Fr. B, B220+ CD43+ HSA+ BP-1−; Fr. C, B220+ CD43+ HSA+ BP-1+), and marginal zone (MZ; B220+ CD21high CD23low) and follicular (FO; B220+ CD21low CD23high) B cells have been sorted by FACS to more than 98% purity. Ebf1 mRNA levels were normalized to β-actin, and the expression level of HSC was set to 1. (F) Quantitative RT-PCR analysis of endogenous levels of Ebf1α and Ebf1β mRNA. The ratio of Ebf1β to Ebf1α mRNA is displayed. Ebf1β mRNA is predominantly expressed in all analyzed cell types, and the ratio increases during pro-B-cell development, with the highest ratio being in fraction C pre-B cells.