Cloning and functional characterisation of avian transcription factor E2A
© Conlon and Meyer; licensee BioMed Central Ltd. 2004
Received: 23 April 2004
Accepted: 14 June 2004
Published: 14 June 2004
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.
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 ). 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 . Binding sites (consensus CANNTG) for these transcription factors are found in all Ig enhancers as well as a number of genes required for heavy and light chain rearrangement (λ5, rag-1, rag-2, EBF). Remarkably, over-expression of E2A together with the recombinase activating genes is sufficient to allow rearrangement of the endogenous Ig locus in a non-lymphoid cell line .
E2A also plays a role in peripheral B cell differentiation. E2A protein expression is greatest in the highly proliferative dark zones of germinal centres, where class switching (CSR) and somatic hypermutation (SHM) are thought to occur. In keeping with such a role, repression of E2A via Id proteins inhibits CSR  and leads to lower expression of the AID (activation induced deaminase) gene , a gene that is essential for both CSR and SHM to occur [8, 9].
Avian B cell development differs from mammalian B cell development in a number of important aspects. Rearrangement occurs between a single VLand JL segment to yield a single functional variable light chain gene. Similarly a single VH segment combines with one of 15 D and a single J segment  generating only a small repertoire. Gene conversion subsequently utilises a pool of upstream pseudo V-genes to generate diversity [10, 11]. Thus gene conversion is the primary mechanism to establish the B cell repertoire in chickens. The diversification and expansion of B cell progenitors occurs in the specialised microenvironment of the Bursa of Fabricus from where mature B cells exit into the periphery. By 6–8 months the diversity of the B cell compartment is established and the bursa involutes. Given the distinct nature of avian B cell development we were interested to find out whether the transcription factor E2A plays a similar central role in B cell ontogeny and the generation of diversity.
To this end we have now cloned full-length avian E12 and E47 cDNAs from the chicken B cell lymphoma DT40 and have initiated their functional characterisation.
Results and Discussion
Cloning of chicken E12 and E47
The resultant full-length E12 and E47 protein sequences were compared to the human, mouse and xenopus orthologues using CLUSTAL W , and the results of this analysis are depicted in Fig. 1. Chicken E12 and E47 are identical except for their bHLH domain, which is nevertheless highly homologous with 72% identity and 86% similarity when allowing only conservative changes. Compared to their human orthologues, chicken E47 and E12 are 71% and 69% identical, respectively. Within the bHLH domains the conservation is even greater. Chicken and human E47 bHLH are 93% identical and chicken and human E12 bHLH are 92% identical. Thus E47 bHLH domains of different species are more similar than the E12 and E47 domain within one species. With regard to the other species examined, chicken E12 is slightly more homologous to xenopus E12 (70%) than to mouse E12 (68%). Outside the bHLH domain there are a number of well-conserved domains. In particular, activation domain I (ADI) is highly homologous across all four species examined (see Fig. 1). The chicken sequence corresponds to the helix consensus described for interaction with the SAGA complex , indicating that E2A mediates some of its function at the chromatin level. Activation domain II has been identified as a domain required to drive transcription in insulin producing β-cells . Within activation domain II only the 5' region is conserved, while insertions are found within the 3' region. However the length of the conserved region, which is significantly longer than that of the ADI, may indicate that this domain has additional functions. The putative nuclear localisation domain is identical in all the compared sequences. Another functional region of the E47 protein is the domain interacting with ubiquitin conjugating enzyme UbcE2A (477-530aa [18, 19]). Within this region, runs of very high homology are seen between the sequences. Whether this is sufficient to obtain functional interaction with UbcE2A awaits experimental confirmation.
In conclusion all regions of very high sequence conservation correspond to functionally important domains.
Genomic structure of the E2A gene
Chicken E2A expression pattern
Functional analysis of chicken E2A
Here we report the cloning of the chicken orthologue of the human transcription factor E2A. The identification of chicken orthologues of genes known to be important for the development of the immune system is of great interest with regard to understanding a related but distinct immune system as is found in birds. One of the advantages of utilising chicken as an experimental system is the existence of the bursal cell line DT40, which undergoes homologous recombination with high frequency thus allowing a genetic approach to the dissection of biochemical pathways. We plan to utilise DT40 in the analysis of E2A function in mature B cells. In mammalian cells, E2A is known to contribute to the regulation of class switch recombination and it has been suggested that it may contribute to SHM either directly or indirectly through controlling AID gene expression. Normally IgV gene diversification in chicken occurs by gene conversion, although somatic mutation can occur . However, cells carrying mutations in the Rad51 homologues XRCC2 and XRCC3 undergo SHM with very high frequency . The identification of avian E2A, together with the recent description of an in vitro somatic mutation assay in DT40 cells, should allow us to investigate the exact role E2A plays in regulating the complex events that confer specificity on the SHM mechanism.
RACE PCR and sequencing
Sequence, 5' to 3'
PCR reactions on serial dilutions of DT40 cDNA prepared using the SMART RACE cDNA Amplification Kit, were carried out using primer pairs P10 and P11 for E12 expression and P10 and P12 for E47 expression. 2 μl of cDNA was used in 20 μl reaction mixtures containing 0.2 mM of each dNTP, 2 μl 10x PCR buffer (Applied Biosystems, UK), 1.5 mM MgCl2 (E12) or 2 mM MgCl2(E47), 1 μM of each primer and 0.5 U Taq Gold (Applied Biosystems, UK). PCR conditions were 94°C for 10 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 3 min, followed by a 10 min extension at 72°C. Products were analysed on a 1.5% agarose gel. A NeverFail Northern Blot (RNway Laboratories) of 2 μg chicken poly A+ RNA per lane was probed with a 32P-labelled fragment of E2A genomic DNA, overlapping the two exons from 1454–1576 and 1577–1726 of the cDNA. The β-actin control probe was generated by PCR using primers P15 and P16.
Full length cDNAs for expression cloning of E12 and E47 were generated by a 2-step PCR reaction using primer pairs P7, P8 (3' end of proteins) and P9, P5 (extending to 5' start). Clones were confirmed by sequencing and cloned into the pcDNA3 expression vector (Invitrogen, USA) using Bam H I and Not I to generate ΔAD1 E12 and E47 constructs. Both E12 and E47 are truncated at amino acic 221 (indicated in bold in figure 1). Subsequently the 5' ends were inserted using Kpn I and Bam H I to generate full length clones. The MDE47 vector was obtained from M. Sigvardsson and contains a forced dimer of hamster E47 in pcDNA . The 6xE2A luciferase vector (pXp2Luc) from A. Green's laboratory contains 3 copies of the following sequence: gtcgaaca gatg ttcacacgacca tctg tgg. pGL3-control and promoter vectors are from Promega, UK. Mouse Id1 was amplified using the primer pairs P13, P14 and cloned into the pCR2.1-TOPO vector. Id1 was subcloned into pcDNA3.1/Hygro expression vector (Invitrogen, USA) using Bam H I and Not I. Full length mouse Id3  was subcloned into the same expression vector using Bam H I and Hind III.
Cell culture and transfection
The chicken bursal B cell line DT40 cells was obtained from Dr J. Sale and maintained in RPMI-1640, 50 μM β-mercaptoethanol, 7% foetal calf serum, 3% chicken serum and antibiotics; human embryonic kidney cells, HEK293, were from Dr K.J. Patel and maintained in DMEM, 10% foetal calf serum and antibiotics. 1 μg each of luciferase reporter and transactivation plasmids, and 0.25 μg of CMV-β-galactosidase vector, were transfected using Superfect Reagent (Qiagen, UK) according to the manufacturer's instructions. We harvested cells 36–48 hours after transfection and generated cell extracts in 200 μl Promega reporter lysis buffer. Luciferase and β-galactosidase activity in 5 μg of protein was measured using Promega reagents. Results are given as ratios of luciferase over β-galactosidase activity.
This work has been funded by the UK Medical Research Council and The Royal Society.
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