Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif

Abstract

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family.

Structure of an Ebf1:DNA complex reveals unusual DNA recognition and structural homology with Rel proteins

“…After completion of this study, the Structural Genomics Consortium (SGC) reported the structures of individual domains of Ebf1 and Ebf3 in the absence of DNA (Siponen et al 2010). The domain structures presented by the SGC are essentially identical to the folds found in the DNAbound dimers of our study.…”

“…The predicted binding to the DNA is highly asymmetric and involves contacts of the Zn knuckle to DNA bases in the major groove, none of which reflects the actual binding mechanism. Finally, Siponen et al (2010) claim that the first two helices of the HLH domain form a new helical bundle-like fold, which is not supported by the obvious similarity with the dimerizing regions of other HLH factors (i.e., MyoD; RMSD 2.7 Å for 40 amino acids). However, the duplicated HLH, which is not visible in our structure, is weakly defined in one monomer of the Ebf3 TIG/HLH structure, and is found to interact with the HLH of the other monomer, suggesting that it may contribute to dimerization.…”

Interaction of CCR4–NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

“…Previous structural analysis of DNA-bound homodimeric EBF1 indicated that the DBD (amino acids 24-240) has a pseudo-Ig-like β-sandwich fold with a structural similarity to the Rel homology domain (Siponen et al 2010;Treiber et al 2010a). DNA binding by EBF1 involves three loops and a zinc knuckle, whereas other loops that connect β sheets or connect the DBD with the IPT domain are potentially available for protein interactions (Treiber et al 2010a).…”

“…EBF1 consists of an extended DNA-binding domain (DBD) with a structural homology with NF-κB, an IPT (Ig-like, plexins, and TFs) domain, an HLH (helix-loop-helix) dimerization domain, and an unstructured C-terminal domain (Siponen et al 2010;Treiber et al 2010a). EBF1 regulates genes by activation and repression and by modulating the chromatin structure (for review, see Hagman et al 2012;Boller and Grosschedl 2014).…”

Molecular mechanisms in lymphoid restriction : securing the B lineage fate

“…EBF1-molecules are thought to bind to DNA as dimers by interacting with two pseudo-palindromic half sites separated by a 2 base pair spacer, ATTCCCNNGGGAAT, where NN can be any nucleotide (Hagman et al, 1991;Travis et al, 1993). The crystal structure of the different domains of EBF1, and also of the DNA-binding domain (DBD, amino acids 26-240) bound to DNA was determined by two groups in 2010 (Siponen et al, 2010;Treiber et al, 2010). Besides its expression in hematopoietic development, EBF1 has a role in adipocyte differentiation (Akerblad et al, 2002) neuron and brain development (Garel et al, 1999;Wang and Reed, 1993) and osteoblast development (Hesslein et al, 2009).…”