Cloning and functional characterisation of avian transcription factor E2A

Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47.The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.The transcription factor E2A contributes to transcriptional regulation in many cell lineages. However, it is essential for the development of B lymphocytes [1,2]. Its role in mammalian B cell development has been studied extensively and E2A functions in B cell commitment and proliferation as well as immunoglobulin (Ig) gene rearrangement and expression (reviewed in [3]). By alternative splicing the E2A gene encodes two basic helix-loop-helix (bHLH) proteins, E12 and E47, which differ only in their highly homologous DNA binding and dimerisation domains [4]. Binding sites (consensus CANNTG) for these transcription factors are found in all Ig enhancers as well as a number of genes required for hea