The Pax-5 Gene Is Alternatively Spliced during B-cell Development

Abstract

The transcription factor Pax-5 is expressed during the early stages of B-cell differentiation and influences the expression of several B-cell-specific genes. In addition to the existing isoform (Pax-5, which we have named Pax5a), we have isolated three new isoforms, Pax-5b, Pax5d, and Pax-5e, from murine spleen and B-lymphoid cell lines using library screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. Isoforms Pax-5d and Pax-5e have deleted the 3 -region, which encodes the transactivating domain, and replaced it with a novel sequence. The existence of alternative Pax-5 transcripts was confirmed using RNase protection assays. Furthermore, Pax-5a and Pax-5b proteins were detected using Western blot analysis. Pax-5a was detectable in pro-, pre-, and mature B-cell lines, but not in two plasmacytomas; Pax-5b was shown to be present at low levels in mature B-cell lines and, unexpectedly, in one plasma cell line, but not in pro-B-cell or T-cell lines. Mobility shift assays showed that in vitro translated Pax-5a and Pax5d, but not Pax-5b or Pax-5e, could interact with a B-cellspecific activator protein-binding site on the blk promoter. Using this assay, we also showed that Pax-5d was present in nuclear extracts of some (but not all) B-lymphoid lines and interacts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isoforms suggests that they may be important regulators of transcription during B-cell maturation.Development of multicellular organisms involves highly regulated expression of specific transcription factors that direct cells into their proper differentiation pathways through interactions with specific target genes. Typically, transcription factors are divided into groups on the basis of the structural and functional similarity of their DNA-binding domains, such as the basic leucine zipper, basic helix-loop-helix, zinc finger, and homeobox families. One small family of transcription factors involved in development is the paired box (Pax) gene family, containing at least nine members, Pax-1 through Pax-9 (1, 2). All members contain a conserved DNA-binding domain encoding the paired box, which consists of a 128-amino acid sequence at the amino-terminal side of the protein. Other conserved regions in Pax proteins include an octamer and a homeodomain sequence in the center of the protein and a transactivating domain that spans 100 amino acids in the Ser/Thr/Pro-rich region at the carboxyl-terminal end (3, 4).Pax genes are grouped into four classes based on structural similarity (5). Class I contains Pax-1 and Pax-9, which lack a homeodomain. Two Class II members, Pax-3 and Pax-7, encode both an octamer and a complete homeodomain. Class III, which includes Pax-2, Pax-5, and Pax-8, represents the class of Pax genes containing an octamer sequence as well as an incomplete homeodomain. Finally, Class IV contains Pax-4 ...

BSAP (Pax5)-Importin α1 (Rch1) Interaction Identifies a Nuclear Localization Sequence

“…1) was generously provided by Stephen Desiderio, Johns Hopkins University, Baltimore, MD (27) (29). Neither the N-terminal paired domain (DNA binding) nor the central domain of BSAP showed autonomous transcriptional activation, and hence both were candidates for detection of protein interactions in the two-hybrid transcriptional activation assay.…”

“…LexA-BSAP Constructs-LexA-BSAP fusion constructs for use in the two-hybrid assay were prepared by cloning PCR fragments amplified from pBSK-BSAP (27) into the EcoRI/SalI sites (underlined) of the pEG202 vector. All constructs were verified by sequencing.…”

Isoforms of the Ets Transcription Factor NERF/ELF-2 Physically Interact with AML1 and Mediate Opposing Effects on AML1-mediated Transcription of the B Cell-specific blk Gene

“…The blk promoter contains a previously uncharacterized NERF/ELF-1-binding site adjacent to a BSAP and AML1 site. Not much is known about regulation of blk gene expression, except that the B cell-specific transcription factor BSAP plays an important role and that the transcription factor NF-B/p50 interacts with the blk gene during B cell activation (41). We furthermore demonstrated that AML1 binds to the blk promoter and cooperatively transactivates the blk promoter in the presence of BSAP (43).…”

“…Hematopoietic genes containing high affinity NERF/ ELF-1-binding sites include, among others, IgH and terminal deoxynucleotidyltransferase (34,35), mb-1 (membrane-bound immunoglobulin IgM-␣) and B29 (36), BSAP (37), lck (38), blk (B lymphoid kinase) (39), and lyn (40,41). Blk is a B cellspecific tyrosine kinase that is expressed in pre-B and mature B cells, but not in plasma cells; this is similar to the expression of mb-1 (membrane-bound immunoglobulin IgM-␣) and B29 (42).…”

PAX5α Enhances the Epithelial Behavior of Human Mammary Carcinoma Cells

“…In addition to the paired box domain, PAX5 contains a partial onehelixed homeodomain and an 8-amino-acid octapeptide motif that functions as a transcriptional inhibitory motif (4). A number of alternatively spliced mRNA transcript variants have been described for both human and mouse PAX5, which result in both NH 2 -terminal and COOHterminal protein variations (10,11). Two developmentally regulated alternative PAX5 splice variants containing distinct 5′-exons (exon 1A and 1B), which encode two isoforms of PAX5 (PAX5α and PAX5β), were originally described by Busslinger et al (11).…”