Emerging roles of the EBF family of transcription factors in tumor suppression

Abstract

Alterations in various developmental pathways are common themes in cancer. The early B-cell factors (EBF) are a family of four highly conserved DNA-binding transcription factors with an atypical zinc-finger and helix-loop-helix motif. They are involved in the differentiation and maturation of several cell lineages including B-progenitor lymphoblasts, neuronal precursors, and osteoblast progenitors. During B-cell development, EBF1 is required for the expression of Pax5, an essential factor for the production of antibody-secreting cells. Accumulating evidence indicates that genomic deletion of the EBF1 gene contributes to the pathogenesis, drug resistance, and relapse of B-progenitor acute lymphoblastic leukemia (ALL). Epigenetic silencing and genomic deletion of the EBF3 locus in chromosome 10q are very frequent in glioblastoma (GBM). Strikingly, the frequency of EBF3 loss in GBM is similar to that of the loss of Pten, a key suppressor of gliomagenesis. Cancer-specific somatic mutations were detected in EBF3 in GBM and in both EBF1 and EBF3 in pancreatic ductal adenocarcinoma. These missense mutations occur in the DNA-binding domain or the conserved IPT/TIG domain, suggesting that they might disrupt the functions of these two proteins. Functional studies revealed that EBF3 represses the expression of genes required for cell proliferation [e.g., cyclins and cyclin-dependent kinases (CDK)] and survival (e.g., Mcl-1 and Daxx) but activates those involved in cell cycle arrest (e.g., p21 and p27), leading to growth suppression and apoptosis. Therefore, EBFs represent new tumor suppressors whose inactivation blocks normal development and contributes to tumorigenesis of diverse types of human cancer.

FIGURE 1. The structural features of EBF family of transcription factors

(A) Shown are the four paralogs (EBF1-4) and their corresponding accession numbers (GenBank or SwissProt databases). Specific domains are shown in shaded or striped boxes. Numbers refer to the position of amino acid residue in each protein. Cancer specific somatic mutations in EBF1 and EBF3 are indicated. The different domains are: DBD, DNA-binding domain; TAD, transactivation domain; COE, the signature sequence of EBF family; ZNF, zinc-finger motif; IPT/TIG (Immunoglobulin-like fold, Plexins, Transcription factors, or Transcription factor Immunoglobin); HLH, helix-loop-helix motif. Note that the ancestral EBF appears to have a typical HLH motif (H1LH2a) that lacks the H2b helix. The H2a and H2b appear to have arisen as a result of an exonic duplication in the vertebrates. (B) The signature zinc-finger motif of the EBF family of proteins in complex with zinc is depicted. The adjacent amino acid residues are also shown. This motif has been designated a zinc knuckle.

FIGURE 2. EBF mediates transcriptional activation and repression

EBF1 binds to DNA as a dimer. Both the HLH motif and the DBD are involved in dimerization. EBF1 and EBF3 have been shown to interact with p300. This interaction is likely to underlie EBF-mediated gene activation through acetylation of core histones by p300 and related coactivator CBP. Several known EBF1 target genes are indicated. EBF1 binds to the two related large zinc finger proteins Zfp423 and Zfp521. The COOH-terminal-most zinc fingers (–30) interact with EBF1, and the N-terminal zinc fingers 2–8 bind to DNA. The Mi-2/NuRD (nucleosome remodeling and deacetylase) complex appears to bind to a conserved NH₂-terminal motif in these two zinc finger proteins. Thus, EBF in conjunction with Zfp423/Zfp521 recruits the Mi-2/NuRD complex for EBF-mediated gene repression. The known components of the Mi-2/NuRD complex are depicted. ZF: zinc finger; MDB: the methyl CpG-binding domain; RbA: retinoblastoma associated protein A (also known as Rbbp4 (p48) and Rbbp7 (p46)); MTA, metastasis associated proteins (MTA1, 2 and 3).

FIGURE 3. EBF1 is a critical component of the B cell pathway

Hematopoietic stem cells (HSC) undergo differentiation mediated by PU.1 and Ikaros transcription factors to give rise to common lymphoid precursors (CLP). EBF1 acts in concert with transcription factors E2A and Pax5 to specify the pro-B cell pathway for the differentiation of CLP. There is extensive crosstalk among these factors to ensure commitment to the pro-B lineage. Factors that are known to suppress EBF1-dependent transcription include Zfp423/Zfp521 and the Mi-2/NuRD complex. For comparison, other lineages derived from CLP or HSC are also shown. CMP: common myeloid precursors; NK: natural killer cells.

FIGURE 4. A potential anti-neoplastic pathway involving EBF3

EBF3 regulates the expression of diverse genes involved in cell growth and proliferation. Specifically, ectopic expression of EBF3 activates the expression of p21 and p27 at early time points, which induce cell cycle arrest. At late time points following EBF3 expression, caspase-3 activation, PARP-1 (poly [ADP-ribose] polymerase 1) cleavage and apoptosis of tumor cells occur. Note that both p21 and p27 are downregulated at late time points. However, whether EBF3 directly mediates the repression of p21 and p27 gene is unclear. Nonetheless, enforced EBF3 expression also leads to marked downregulation of cyclins and CDKs as well as proteins involved in cell survival such as Daxx and Mcl-1 at earlier time points. Thus, EBF3 might directly repress these genes.