Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes

Abstract

Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

FIG. 1

ABF-1 is capable of interacting with the E proteins in the two-hybrid system. Expression plasmids encoding the indicated GAL4 DNA binding domain fusions (E-protein bait) and GAL4 AD fusions were cotransformed into the reporter yeast strain y190, which contains an integrated GAL1-HIS3 reporter gene (19). Transformants were initially selected on synthetic medium lacking tryptophan and leucine and then spotted onto synthetic medium lacking histidine and containing 25 mM 3AT. Growth on the selective medium indicates an interaction between the E-protein bait and the GAL AD fusion protein (19). Yeast cells were allowed to grow for 4 days at 30°C and then photographed. Vector, pACT2; CL.95, clone 95; ABF-1, pACT2-ABF-1; ID2, pACT-ID2. The GAL4 AD–ABF-1(FL) fusion conferred a mild slow-growth phenotype in yeast strain y190.

FIG. 2

Nucleotide sequence of the human ABF-1 cDNA. The longest open reading frame encodes a protein 218 amino acids in length. Although not full length (see Fig. 4), the ∼1.9-kb cDNA contains two potential start codons, both with good Kozak sequences (58). The conceptual translation product predicts a 23.6-kDa protein with an estimated pI of 9.5. In addition to the bHLH motif (bold underline), there is a putative nuclear localization signal (dashed underline), a glycine-rich region (boldface), and a stretch of acidic residues (double underlined). The boxed leucine residue (position 130) represents the fusion point of clone 95 with the GAL4 AD. The asterisk denotes the stop codon.

FIG. 3

Sequence alignment of ABF-1 with the class II family of bHLH proteins. (A) Multiple sequence alignment of the bHLH region of human (h.) ABF-1 with the most related class II bHLH proteins was created by using the PileUp and Pretty algorithms (Genetics Computer Group sequence analysis software package). Amino acids identical in at least half of the sequences are shown as blackened boxes. For reference, the bHLH regions of the class I proteins E12 and E47 are shown. m., mouse; D., Drosophila; C., Caenorhabditis elegans. (B) Dendrogram displaying a graphical output of the pairwise alignments of ABF-1 and related bHLH family members generated by PileUp.

FIG. 4

Northern blot analysis of ABF-1 expression. (A) Expression pattern of ABF-1 in human cell lines. Ten micrograms of total RNA was isolated from each cell line and analyzed by Northern blotting. The blot was probed with the ABF-1 cDNA (top), stripped, and subsequently reprobed with the human elongation factor 1 alpha (EF-1α) cDNA (6) as a loading control (bottom). HeLa, carcinoma; Jurkat, T-cell leukemia; 697, pre-B ALL harboring a t(1;19) translocation; Nalm-6, pre-B; BL, Burkitt lymphoma; LCL, lymphoblastoid cell line of the indicated Ig isotype. The ER/EB2-5 cell line was grown in the presence of 1 μM β-estradiol (27). (B) A human tissue Northern blot (Clontech) containing 2 μg of poly(A)⁺ RNA per lane was sequentially hybridized with ABF-1 (top) and β-actin (bottom). PBL, peripheral blood lymphocyte.

FIG. 5

ABF-1 mRNA is highly abundant in activated human B cells. Northern blot analysis of 10 μg of total RNA isolated from purified peripheral blood B lymphocytes activated in vitro. B cells were treated with SAC for 3 days or with SAC and IL-2 for 6 days or were left untreated. The Northern blot was probed with the ABF-1 cDNA (top), stripped, and then reprobed with elongation factor 1 alpha (EF-1α; bottom) as a control.

FIG. 6

ABF-1 binds to an E box in vitro. (A) EMSA analysis of FLAG ABF-1 homo- and heterodimeric complexes formed on the μE4-OCT probe. Arrows indicate the position of ABF-1 homodimers and ABF-1–E2A heterodimers. retic., reticulocyte lysate; FP, free probe; NS, nonspecific complex; S, supershifted complex. (B) ABF-1 binds as a heterodimer to the μE4 probe. Addition of a nonspecific control antibody had no effect on complex formation (not shown).

FIG. 7

Western blot analysis of ABF-1 in nuclear extracts isolated from several human cell lines. Lane 1, EBV-immortalized LCL B3C1; lane 2, B5D5 (LCL); lane 3, Namalwa (Burkitt lymphoma); lane 4, DLD1 (colon carcinoma). ABF-1 was detected by using a polyclonal antibody as described in Materials and Methods.

FIG. 8

ABF-1 is part of an E-box binding complex present in EBV-immortalized LCLs. (A) Gel shift analysis with a μE5 probe reveals a novel complex (N) whose mobility differs from that of BCF present in the EBV-immortalized line B3C1 (lane 2). (B) The ABF-1-containing nucleoprotein complex, indicated by arrows, was detected in all EBV-immortalized LCLs (seven cell lines in total were analyzed). Lane 1, unprogrammed reticulocyte lysate; lanes 2 to 5, in vitro-cotranslated ABF-1 and E12 proteins; lanes 6 to 11, nuclear extract derived from the conditional cell line ER/EB2-5 (32) grown in the presence of 1 μM β-estradiol (+est) or estrogen starved for 48 h (−est); lanes 12 to 26, nuclear extract isolated from independent LCLs. N, no antibody; P, preimmune serum; A, ABF-1-specific antiserum. The free probe was run off the gel.

FIG. 9

ABF-1 functions as a transcriptional repressor in mammalian cells. (A) ABF-1 interferes with the ability of E47 to activate transcription in mammalian cells. HeLa S3 cells were transiently transfected with 1 μg of (μE5-μE2)₆CAT reporter plasmid, 0.25 μg of E47 expression vector (pHβA-E47), and increasing amounts (shown in micrograms) of empty expression vector (pHβAneo) or FLAG–ABF-1 expression vector (pHβA-FLAG-ABF-1) as indicated. The total amount of DNA used in each transfection experiment was adjusted to 5 μg by addition of pBSK; 30 μl of Superfect reagent was used for each transfection. Equal amounts of protein extracts were assayed for CAT activity. CAT activity is indicated by % acetylated, which represents the conversion of ¹⁴C-chloramphenicol to acetylated forms. (B) ABF-1 contains a transcriptional repression domain. HeLa S3 cells were transiently transfected as described above with 1 μg of 3XUAS_GALTK LUC reporter plasmid, 0.025 μg of pCMVβGAL, and 1 μg of the indicated GAL4 expression constructs. In the absence of effector plasmid, the 3XUAS_GALTK LUC reporter gene activity was 16,546 relative light units. Luciferase activity was normalized to β-galactosidase levels as a control for transfection efficiency.