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BMC Immunol
2004 Jun 14;5:11. doi: 10.1186/1471-2172-5-11.
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Cloning and functional characterisation of avian transcription factor E2A.
Conlon TM
,
Meyer KB
.
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During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of 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.
Figure 1. Alignment of E2A sequences from human, mouse, xenopus and chicken: full-length E12 (A) and the E47 bHLH exon (B) which replaces the E12 bHLH exon in the full-length sequence. "*" denotes identical residues, ":" conserved substitutions as recorded in CLUSTAL, and "." semi-conservative substitutions. Accession numbers for the sequences used are: PI5923, XM125750, X66959 for human, mouse and Xenopus, respectively. The chicken sequence has been assigned the accession number AJ579995 for E12 and AJ579996 for E47. Shaded boxes denote functionally defined regions: activation domain I (ADI) with boxed helix [29], activation domain II (ADII) [17] and the ubiquitin ligase interaction domain [18]. The putative NLS is underlined and the bHLH region is boxed in black. The exon border of the xenopus sequence is based on its homology to the other sequences. Chicken amino acid 221 (bold) is the first aa of the ÎADI constructs used in the functional analysis.
Figure 2. Schematic representation of the structure of the chicken and human E2A genes. Black lines represent assigned exons, grey lines represent exons lying in gap sequences; their exact position has not been determined. Open boxes represent the alternatively spliced E12 and E47 exons encoding the helix-loop-helix domain of these proteins. See Table 2 for the exact position of each of the exons.
Figure 3. A. PCR analysis of E12 and E47 on DT40 cDNA using increasing dilutions of cDNA. M, DNA markers. B. Northern Blot of E2A expression, with 2 μg polyA+ RNA from the tissues shown above the lanes. A β-actin probe was used as a control.
Figure 4. Transcriptional activation by mammalian E47 and chicken E12 and E47 in HEK293 cells. A. Transactivation of a multmerised E2A binding site by different E2A proteins as indicated. Values are given relative to those obtained with the pcDNA control vector. The data shows the mean +/- SEM of triplicate transfections. B. Effect of co-transfection of Id expression constructs on E47 transcriptional activation. The data show the mean +/- SEM of five independent transfections.
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