Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin (Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites—known to be important for the transcriptional role of Ig enhancers—impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.
During the B cell immune response, immunoglobulin (Ig) genes are subject to a unique mutation process known as somatic hypermutation that allows the immune system to generate high-affinity antibodies. Somatic hypermutation preferentially affects Ig genes, relative to other genes, and this is important in preventing catastrophic levels of general genomic mutations that could lead to B cell cancers. We hypothesized that this preferential targeting of somatic hypermutation is assisted by specific DNA sequences in or near Ig genes that focus the action of the mutation machinery on those genes. In this study, we show that Ig genes across species—from human, mouse, and chicken—do indeed contain such mutation targeting sequences and that they coincide with transcriptional regulatory regions known as enhancers. We show that combinations of Ig enhancers cooperate to achieve strong mutation targeting and that this action depends on well-known transcription factor binding sites in these enhancer elements. Our findings establish an evolutionarily conserved function for enhancers in somatic hypermutation targeting, which operates by a mechanism distinct from the conventional enhancer function of increasing levels of transcription. We propose that combinations of Ig enhancers target somatic mutation to Ig genes by recruiting the mutation machinery and/or by making the Ig genes better substrates for mutation.
Citation: Buerstedde J-M, Alinikula J, Arakawa H, McDonald JJ, Schatz DG (2014) Targeting Of Somatic Hypermutation By immunoglobulin Enhancer And Enhancer-Like Sequences. PLoS Biol 12(4): e1001831. doi:10.1371/journal.pbio.1001831
Academic Editor: David Nemazee, Scripps Research Institute, United States of America
Received: December 11, 2013; Accepted: February 21, 2014; Published: April 1, 2014
Copyright: © 2014 Buerstedde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: J-M.B. was supported by an International Outgoing Marie Curie Fellowship. J.J.M. was supported in part by NIH T32 AI07019. D.G.S is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: GCV, immunoglobulin gene conversion; SH, somatic hypermutation
The appearance of point mutations within the rearranged immunoglobulin (Ig) genes of B cells, which leads eventually to the selection and production of high-affinity antibodies, is called somatic hypermutation (SH) ,. SH requires transcription of the Ig genes  and expression of the activation-induced cytidine deaminase (AID) protein encoded by the AICDA gene ,. AID is believed to initiate all three types of B cell–specific Ig gene diversification—SH, Ig gene conversion (GCV), and Ig class switch recombination—by deaminating cytidines within the Ig loci –.
While many non-Ig genes accrue mutations in AID-expressing B cells as a result of SH, Ig genes mutate at levels that are typically several orders of magnitude greater than those of non-Ig genes –. The question of how SH is preferentially targeted to Ig loci has been studied and debated for over 20 years. Pioneering experiments using chimeric gene constructs in transgenic mice indicated that sequences overlapping with the Ig light chain and Ig heavy chain enhancers distinguish the Ig genes as mutation targets –. Other early transgene studies indicated that Ig V region sequences themselves are not required for SH  and that active heterologous promoters can support SH ,. However, further insight into the nature of the putative cis-acting regulatory elements was hampered by the laborious transgene experimental system, the relatively low mutation rates of the chimeric genes, and the fluctuation of mutation rates among transgenic lines, perhaps due to integration site effects and copy number variations. A further problem arose from the fact that the putative hypermutation-stimulating sequences included the known enhancers, making it difficult to differentiate between the effects of these sequences on transgene hypermutation versus transgene transcription (reviewed in ).
The hypothesis that SH is targeted preferentially to Ig genes by the Ig enhancers was subsequently called into question when germline deletions of individual murine Ig enhancers—the same sequences previously implicated in the hypermutation of chimeric transgenes—did not abolish SH within the respective loci –. It also became apparent that expression of either AID or the related cytidine deaminases APOBEC-3A or APOBEC-3B increased mutation frequencies in the genomes of fibroblasts , Escherichia coli , yeast , and human breast cancer cells . These findings and others (reviewed in ,) raised widespread doubts about the relevance of specific cis-acting SH targeting elements in Ig loci. In particular, Ig enhancers were no longer regarded as likely SH targeting elements, and it was increasingly felt that they increased SH solely by increasing Ig gene transcription. Attention has recently focused on RNA polymerase II (Pol II)–associated factors that interact with AID and play roles in transcriptional stalling  and RNA processing , processes that are likely to be critical for generating the single strand DNA substrate required by AID (reviewed in ,). However, these broadly acting factors do not provide a ready explanation for the strong preference that SH exhibits for Ig genes over non-Ig genes. Consequently, this has remained a central unresolved issue in the field.
The chicken B cell line DT40, whose genome is easily modified by targeted gene integration , is a powerful model to investigate AID-mediated gene diversification . DT40 variegates its rearranged Ig light chain (cIgλ) gene primarily by GCV , but diversification occurs by SH if either upstream GCV donor sequences or uracil DNA glycosylase (UNG) are missing ,. Evidence for the stimulation of cIgλ GCV by cis-acting sequences in DT40 has been detected by the analysis of endogenous cIgλ gene diversification , transgene GCV , and transgene hypermutation . Reminiscent of the early experiments in transgenic mice, SH of a green fluorescent protein (GFP) knock-in transgene in DT40 cells depended on the nearby presence of a 10-kb fragment of the cIgλ locus, which was named diversification activator (DIVAC) . Deletion analysis of DIVAC led to the identification of two core regions downstream of the cIgλ C-region that cooperate with each other and with other parts of the 10-kb sequence to stimulate SH of the adjacent GFP transcription unit . However, a clearer definition of the DIVAC code proved challenging using the original GFP assay because of functional redundancy within the 10-kb sequence and difficulty in measuring the DIVAC activity of elements shorter than 500 bp –. Furthermore, murine Ig lambda (Igλ) and Ig kappa (Igκ) enhancer sequences displayed disappointingly low DIVAC activity in DT40 cells ,. Hence, the identity of key SH targeting sequences and the extent to which these sequences have been conserved during vertebrate evolution have remained undetermined.
We have now developed a highly sensitive assay that allows analysis of the SH targeting activity of small DNA elements, largely overcoming the shortcomings of previous experimental strategies. Using this new assay, we demonstrate that chicken, mouse, and human Ig locus enhancers and enhancer-like elements are core DIVAC sequences that work together to target SH. Regardless of which species they derive from, these elements rely for function on a common set of well-characterized transcription factor binding motifs, highlighting the evolutionary conservation of the SH targeting mechanism. These findings are likely to have implications for the mistargeting of SH to non-Ig genes and the origins of B cell lymphoma.
A Highly Sensitive DIVAC Assay
We previously developed an assay for DIVAC function that made use of a reporter cassette, termed GFP2, consisting of a strong viral promoter driving expression of GFP and a drug resistance gene (Figure 1A) . In this assay, GFP2, with or without a flanking test sequence, was inserted by homologous recombination into the DT40 genome, and GFP expression was monitored in subclones by flow cytometry. Loss of GFP expression was entirely dependent on AID, was due to point mutations in GFP, and could be stimulated more than 100-fold by the presence of a strong DIVAC element adjacent to the GFP2 cassette . Importantly, three previous studies demonstrated that DIVAC-dependent stimulation of GFP mutation was not accompanied by substantial changes in GFP transcription as measured by several methods, demonstrating that DIVAC stimulates SH by a mechanism independent of an increase in transcription –.
(A) Diagram of the GFP2 and GFP4 hypermutation reporters. RSV, rous sarcoma virus; bsr, blasticidin resistance gene; IRES, internal ribosome entry site; ATG, the start codon of the hypermutation target sequence–GFP fusion protein encoded by GFP4. (B) Map of the rearranged chicken Igλ (cIgλ) locus with the location of sequences conserved among avian species indicated by rectangles. Sequences tested are shown below, with their orientation relative to GFP4 indicated by arrows. VJ, rearranged variable region gene; C, constant region; E, enhancer. (C) Flow cytometry profiles of representative subclones of primary transfectants carrying either GFP4 alone (UNG−/−AIDR) or GFP4 combined with the sequence specified above each plot. All transfectants are UNG-deficient, AID-reconstituted, except UNG−/−AID−cIgλE↔3′Core, which does not express AID. (D) Graph showing the percent GFP loss of individual subclones. Each dot represents a subclone. The median GFP loss for each group of subclones is indicated by the bar and numerically displayed above the graph.
To increase the sensitivity of the DIVAC assay, we modified the GFP2 reporter by the insertion of a 5′ untranslated sequence upstream of the methionine start codon and a hypermutation target sequence between the start codon and the GFP open reading frame, yielding the new reporter GFP4 (Figure 1A). The 249-bp hypermutation target sequence consists of repetitions of TGG, CAA, and CAG codons frequently positioned in the context of SH hotspot motifs WRCY/RGYW (W = A or T; R = A or G; Y = C or T). Transition mutations at the second or third position of the TGG codons or at the first position of the CAA and CAG codons will introduce nonsense mutations, precluding the translation of the GFP open reading frame (Figure S1).
To further increase the frequency at which mutations and stop codons are generated, the GFP4 assay is performed in UNG-deficient cells, which accumulate exclusively C-to-T and G-to-A transition mutations and display a 7-fold increased rate of SH , most likely because AID-induced uracils cannot be excised and repaired before replication. To assay DIVAC-GFP4 combinations at a defined chromosomal position, we generated a recipient cell line, UNG−/−AIDR/puro, in which (i) both endogenous UNG genes were disrupted and the coding sequences of both endogenous AICDA genes were deleted, (ii) AID expression was reconstituted by inserting an AICDA cDNA expression cassette under the influence of the β-actin promoter into one AICDA locus, and (iii) the position of the second AICDA locus was marked by a puromycin resistance gene. When this cell line is transfected by AICDA locus–targeting constructs containing DIVAC-GFP4, targeted integrants into the marked AICDA locus are easily identified by the loss of puromycin resistance.
Conservation of DIVAC Sequences
Alignment of the cIgλ locus with the corresponding sequence of turkey, zebra finch, and ground finch revealed seven evolutionarily conserved sequence contigs downstream of the C-region (Figures 1B and S2). Two of these corresponded closely to regions we had previously demonstrated to be important for DIVAC function in the context of larger DNA elements : the cIgλ enhancer (cIgλE)  and the 3′Core. The conserved sequence regions were cloned into the upstream DIVAC insertion site of GFP4 (the default site used in all experiments except where indicated) and transfected into UNG−/−AIDR/puro cells. Primary transfectants with targeted integration of a construct were subcloned, and 24 subclones were analyzed for GFP loss by flow cytometry 12 d after subcloning (Figure 1C and 1D). Transfectants containing cIgλE or 3′Core, in either orientation (reverse orientation indicated by “R”), showed median GFP loss levels of 20%–30%, whereas levels of GFP loss in transfectants of the other conserved sequences (Con1–Con5) were close to the 1.7% median value observed in the no DIVAC control transfectant, UNG−/−AIDR. Interestingly, the Con2 sequence, which displayed activity close to background on its own, substantially increased GFP loss when combined with cIgλE in Con2+cIgλE cells (44.6%). The highest levels of GFP loss were seen when cIgλE and the 3′Core were combined (63.7%) or when they were tested together with their intervening sequence (cIgλE↔3′Core; 70.5%). Importantly, GFP loss in UNG−/−AID−/−cIgλE↔3′Core cells (lacking the AICDA expression cassette) was almost 3,000-fold lower than in cIgλE↔3′Core cells and about 60-fold lower than in UNG−/−AIDR cells.
These results illustrate several points. First, the DIVAC-GFP4 assay is capable of detecting robust stimulation of SH by short DNA fragments, which heretofore has not been possible. Second, these results directly confirm the role of cIgλE and 3′Core as core DIVAC elements . Third, in the absence of DIVAC, GFP loss from GFP4 in UNG−/− cells is 15- to 20-fold higher than we detect with GFP2 in wild-type cells (see below, and ,), likely reflecting both the increased sensitivity of GFP4 and an increase of DIVAC-independent mutations in the UNG-deficient background. Finally, in the absence of AID, UNG deficiency does not lead to substantial GFP loss, even in the presence of a strong DIVAC element. Hence, despite the repair-deficient context, both DIVAC-dependent and DIVAC-independent GFP loss in the GFP4 assay require AID.
Sequencing of the hypermutation target region amplified from cIgλE↔3′Core cells 6 wk after subcloning revealed frequent transition mutations at G/C bases with a hotspot preference as expected for SH in UNG-deficient DT40 cells (Figure S1). Many of these mutations yielded stop codons, explaining the efficient GFP loss seen in cIgλE↔3′Core cells.
DIVAC Elements Require Transcription Factor Binding Motifs
cIgλE includes an E-box as well as NFκB (nuclear factor kappa B), MEF2 (myocyte-specific enhancer factor 2), and PU.1-IRF4 (interferon regulatory family-4) binding motifs, all of which are remarkably conserved among avian species (Figure S2B). Deletions starting either from the 5′ or the 3′ end of cIgλE progressively decreased GFP loss in the DIVAC assay (Figure 2A and 2B). Once the 5′ deletions reached the NFκB motif (5′Δ37), GFP loss fell to background levels. Similarly, 3′ end deletions including the IRF4 motif in 3′Δ49 cells strongly reduced GFP loss.
(A) Diagram of the cIgλE fragment with truncations indicated below and conserved transcription factor binding motifs depicted as rectangles. The sequences of binding sites and binding site mutants are shown on the right. (B) GFP loss of subclones in the presence of full-length, truncated, and mutated cIgλE sequences.
The role of specific binding site motifs was further investigated by mutation of consensus residues in these sites (Figure 2A and 2C). Whereas mutations in the NFκB, MEF2, PU.1, or IRF4 motifs strongly decreased GFP loss, mutations in the E-box caused a more modest reduction, and a mutation in the spacer between the PU.1 and IRF4 motifs was well tolerated (Figure 2C). These results indicate that cIgλE requires the integrity of multiple transcription factor binding sites in its 5′ and 3′ halves for full activity.
Little was known about 3′Core, the second autonomous DIVAC sequence of the chicken Igλ locus. Deletion of the first 42 and the last 99 bp did not affect GFP loss (5′Δ42_3′Δ99), whereas many deletions in the central part of the fragment reduced GFP loss (Figure S3A and S3B). Search algorithms for transcription factor binding motifs predicted, among others, six evolutionarily conserved binding motifs in the parts of 3′Core where deletions compromised activity: three E-boxes and three other putative sites, referred to as pCBF (core binding factor), pC/EBP (CCAAT enhancer binding protein), and pPU.1 (Figure S2C) (where “p” designates a putative binding site for which experimental evidence linking it to the factor is lacking). Deletion or mutation of any one of these motifs, with the exception of pPU.1, reduced GFP loss substantially, with the strongest effects seen for E-box2, pCBF, and pC/EBP, which lie close together in the central part of the fragment (Figure S3C and S3D). Thus, evolutionarily conserved transcription factor binding motifs are also critical for the DIVAC function of 3′Core. We note that many more sites were predicted in silico than were tested, and the factors that might bind to these and the tested sites, particularly pCBF and pC/EBP, remain unknown.
The Human Igλ Enhancer as a Surprisingly Strong DIVAC
Alignment of human, murine, and chicken Igλ enhancer sequences revealed striking conservation of the E-box and NFκB, MEF2, PU.1, and IRF4 binding motifs ,, while the mammalian sequences possess an additional E-box about 50 bp downstream of the PU.1 site (Figure S4A). Since the conserved transcription factor binding motifs were important for the DIVAC function of cIgλE, we reasoned that the mammalian enhancers might also be active DIVAC elements despite low sequence conservation of the intervening sequences.
We began by testing the human Igλ enhancer (hIgλE) in either the upstream or downstream insertion site of GFP4 (Figure 1A), which yielded a remarkable 46% GFP loss (Figure 3B), almost twice the activity of cIgλE (27.2%). Removal of the upstream E-box in 5′Δ56 did not decrease DIVAC activity, whereas larger 5′ deletions reduced activity (Figure 3A and 3B). However, even after removal of the upstream E-box, NFκB, and MEF2 sites, the 5′Δ84 fragment was still capable of supporting 23.6% GFP loss, almost as high as the activity of full-length cIgλE and much higher than the activity of the comparable deletion fragment (5′Δ59) of cIgλE (Figure 2B). These results suggest that the 3′ portion of hIgλE contains important elements and that the downstream E-box might compensate for loss of the upstream E-box-NFκB-MEF2 sites. Consistent with this, a 3′ deletion including the downstream E-box (3′Δ46) reduced GFP loss to 20%—roughly the activity of full-length cIgλE—and a larger 3′ deletion removing the composite PU.1-IRF4 site (3′Δ108) strongly reduced GFP loss to 6% (Figure 3B), similar to the low activity of the comparable cIgλE 3′Δ68 fragment (Figure 2B). In the strongly active hIgλE, point mutations in individual motifs reduced activity, although typically less than 2-fold, and only mutation of both components of the composite PU.1-IRF4 site had a strong effect on activity (Figure 3A and 3B). Therefore, hIgλE is both more active and apparently more robust than cIgλE, being less sensitive to mutation of individual motifs. The major difference between the human and chicken enhancers appears to lie in sequences in their 3′ portions.
(A) Diagram of the hIgλE fragment with truncations indicated below and conserved transcription factor binding sites depicted as rectangles. The sequences of binding sites and binding site mutants are shown on the right. (B) GFP loss of subclones in the presence of full-length, truncated, and mutated hIgλE. hIgλEDown cells carry hIgλE downstream of GFP4.
These results demonstrate, to our knowledge for the first time, a substantial conservation of DIVAC function from human to chicken sequences. They also reveal parallels between the enhancement of SH and the enhancement of transcription by the Igλ enhancer because the transcription factor binding sites long known to be important for the regulation of Igλ transcription – are also critical for DIVAC function.
Enhancers as DIVAC Elements in Mammalian Igh and Igκ Loci
Sequence homologues of mammalian Ig heavy chain intron enhancers (IgHEi) could not be identified in birds, and an enhancer in the intron between the duck Jμ and Cμ segments showed no obvious conservation with mammalian counterparts apart from the presence of multiple E-boxes . Human (hIgHEi) and murine (mIgHEi) enhancer fragments contain conserved YY1 (yin yang 1) (μE1), E-box (μE2 and μE4), Ets1 (μA), PU.1 (μB), IRF, and Octamer transcription factor binding sites, and less well conserved regions μE5 and μE3 , (Figure 4A and S4B). Since these sites overlap substantially with those important for DIVAC function in cIgλE, 3′Core, and hIgλE, we reasoned that the mammalian IgHEi elements might also have SH targeting activity. Strikingly, hIgHEi and mIgHEi yielded high levels of GFP loss (62.1% and 47.3%, respectively; Figure 4B), well above that of cIgλE and 3′Core, and similar to that observed with hIgλE.
(A) Diagram of the hIgHEi fragment with truncations indicated below and transcription factor binding sites depicted as rectangles. The sequences of binding sites and binding site mutants are shown on the right. (B) GFP loss in the presence of hIgHEi, mIgHEi, or truncated or mutated versions of hIgHEi.
To investigate the role of the well-known binding sites, hIgHEi was subject to deletion and mutation analysis. Whereas a 5′ deletion of hIgHEi including the μE1, μE2, μA, and μB sites only moderately decreased GFP loss in 5′Δ109 and 5′Δ136 cells, 3′ deletions including the Octamer, μE4, and IRF sites strongly decreased GFP loss in 3′Δ67 and 3′Δ136 cells. Consistent with the importance of the 3′ part of hIgHEi, mutations of either the μE4 or IRF site strongly decreased GFP loss, whereas an Octamer site mutation had little effect. Thus, the binding sites in the 5′ portion, although able to boost activity of the 3′ portion, are unable to compensate for loss of the IRF or μE4 sites in the 3′ portion. We conclude that mammalian IgHEi sequences are potent DIVAC elements in chicken cells.
Homologues of the mammalian Ig kappa chain (Igκ) enhancers are also not present in avian species, which contain only a single Igλ light chain locus. The three Igκ enhancers, intron (IgκEi), 3′ (IgκE3′), and Ed (IgκEd) , of mice and humans (Figures 5A and S5) induced low or modest levels of GFP loss when assayed on their own (Figure 5B), consistent with previous analyses ,. However, when two Igκ enhancers were combined (IgκEi+IgκE3′ or IgκE3′+IgκEd), GFP loss markedly increased, and when the three human Igκ enhancers were combined, GFP loss reached 50.9% (Figure 5B). This shows that the known synergy of the Igκ enhancers with respect to the activation of Igκ transcription ( and references therein) also holds true for their DIVAC function, even in an avian B cell line lacking an endogenous Igκ locus.
(A) Map of the human Igκ locus showing the locations of the three Igκ enhancers as open rectangles. V, variable gene segment; J, joining gene segment; C, constant region. (B) GFP loss in the presence of individual human and murine Igκ enhancers and enhancer combinations.
Congruence between the GFP4 and GFP2 Assays
To confirm our results in a repair-proficient cellular context (UNG-proficient DT40 cells) and in a different genomic integration site (the deleted rearranged Igλ locus), we tested various cIgλ DIVAC elements using the GFP2 assay. The full cIgλ DIVAC region (the 9.8-kb W fragment that includes the rearranged VJλ region and all downstream cIgλ sequences) yielded about 10% GFP loss using GFP2 (Figure S6B and S6C), consistent with our previous study . In general, the rank order of activities of DIVAC elements was similar between the GFP2 and GFP4 assays (compare Figures S6C and 1D). Comparison of median GFP loss levels indicated that the GFP2 assay is approximately 20–50 times less sensitive than the GFP4 assay (e.g., for cIgλE and 3′Core, respectively: 27.2% and 33.2% median GFP loss with GFP4, and 0.54% and 0.75% median GFP loss with GFP2). However, with the cIgλE↔3′Core fragment, GFP loss in the GFP2 assay (6.7%) was only about 10-fold lower than in the GFP4 assay (70.5%), probably because of saturation of the GFP4 assay in the presence of this highly active DIVAC element (see Protocol S1). We also used the GFP2 assay to confirm that Con2 (which lacks activity on its own) was able to substantially boost the activity of cIgλE (Figure S6C). A limited deletion and mutation analysis of Con2 (Figure S6A) using the GFP2 assay (Figure S6C) and the GFP4 assay (Figure S6D) demonstrated that functional cooperation between Con2 and cIgλE required only the 3′ portion of Con2 and was dependent on one of the two putative IRF binding motifs (pIRF-down) in this region. We conclude that there is good congruence between the results of the GFP4 and GFP2 assays and that the less sensitive GFP2 assay is preferable for analysis of highly active DIVAC elements.
Newly Identified Shadow Enhancers Act as Strong DIVAC Elements in the Murine Igλ Locus
The murine Igλ locus contains two enhancers, mIgλE3-1 and mIgλE2-4, due to a duplication of a pair of J-C regions and their downstream enhancer (Figure 6A) . These enhancers are relatively weak DIVAC elements on their own (0.4%–0.5% GFP loss in the GFP2 assay; Figure 6B), consistent with our previous analysis . This suggested the need for other sequences in the locus to cooperate with mIgλE3-1 and mIgλE2-4 to support efficient SH of murine Igλ (note that cooperation between mIgλE3-1 and mIgλE2-4 is not possible in some rearranged Igλ loci because rearrangement of upstream V2 or V3 gene segments to the JC3 or JC1 clusters deletes mIgλE2-4). However, the identity of such putative cooperating elements was unclear because other murine Igλ enhancers were not known.
(A) Map of the murine Igλ locus showing the locations of the known and newly discovered shadow enhancers (arrows). V, variable gene segment; J, joining gene segment; C, constant region. (B) GFP loss in the presence of individual murine Igλ enhancers and canonical enhancer–shadow enhancer combinations (GFP2 assay). (C) GFP loss in the presence of mammalian enhancers as single copy, multimers, or combinations (GFP2 assay). In (B) and (C), subclones from two independent primary transfectants for each construct were analyzed, as indicated above each plot.
Intriguingly, BLAST searches revealed the presence of IgλE homologues 20–25 kb downstream of mIgλE3-1 and mIgλE2-4 (Figure 6A), which we refer to as mIgλE3-1s and mIgλE2-4s because of their resemblance to shadow enhancers . The newly identified elements are 95% identical to one another and about 70% identical to the canonical enhancers, with the conservation including many of the transcription factor binding motifs shown to be important for DIVAC function of the chicken and human Igλ enhancers (Figure S4A). When tested for DIVAC function, mIgλE3-1s and mIgλE2-4s were substantially more active than the canonical enhancers in both the GFP2 (Figure 6B) and GFP4 assays (data not shown). Strikingly, the combination of a shadow enhancer with its neighboring canonical enhancer induced GFP loss strongly and synergistically (Figure 6B), in the case of mIgλE2-4 plus mIgλE2-4s to levels almost as high as that seen for the entire cIgλ W fragment. These results reveal that strong SH targeting elements can be constructed from combinations of enhancers and enhancer-like elements in the murine Igλ locus, as is true also for chicken Igλ. Furthermore, they demonstrate our ability to identify strong DIVAC elements in the murine Igλ locus on the assumption that Igλ enhancer-like sequences activate SH.
We extended this by investigating the activity of other combinations of elements, continuing to use the GFP2 assay. Consistent with the GFP4 data, hIgλE, hIgHEi, and the combined murine Igκ enhancers supported levels of GFP loss that were more than 20-fold above the background of AIDR cells (0.1%), whereas the 5′Δ84 deletion mutant of hIgλE was less active (Figure 6C). Duplication of the truncated 5′Δ84 or the full-length hIgλE increased levels of GFP loss from about 0.6% and 2.4% to about 2.0% and 6%, respectively, showing that even the interaction between identical sequences can lead to a synergistic increase of DIVAC function, similar to the well-known effects of multimerization of enhancer sequences on transcriptional activity .
DIVAC Elements Have Little Effect on Transcription of GFP4
Consistent with previous studies of the GFP2 reporter , or modifications thereof , mRNA levels from GFP4 were either not significantly or only marginally (up to 2-fold) increased by the presence of chicken or mammalian DIVAC fragments compared to the no DIVAC control (Figure 7A–7C). Therefore, as with the GFP2 assay, DIVAC elements stimulate mutation in the GFP4 assay by a mechanism that is independent of an increase in GFP transcription.
(A) Diagram of GFP4 (the RSV promoter is not shown) showing location of amplicons for the reverse transcription quantitative PCR analyses and the GFP probe used for the Northern blot. (B) Relative GFP mRNA levels in GFP4 transfectants as determined by quantitative PCR. GFP transcript levels were normalized to 18S rRNA levels. Note that the AICDA expression cassette was deleted from all of the lines assayed for GFP transcript levels to avoid effects of nonsense-mediated decay (see Materials and Methods). Data are presented as the mean (± standard error of the mean) of three independent experiments, except for the hIgλE 5′ amplicon (2 experiments). Two-tailed unpaired t-tests were used to compare the value for each DIVAC element to that of the no DIVAC control. *p<0.05; **p<0.01. (C) Northern blot analysis of GFP4 transfectants assayed in (B) and two GFP2 cell lines. Top, hybridization with a GFP probe. Arrows indicate the larger GFP4 and smaller GFP2 mRNA bands; the size difference is as expected from the addition of the hypermutation target sequence to GFP4. Bottom, hybridization of the same blot with a control GAPDH probe. Numbers below each lane indicate GFP mRNA levels after normalization to the GAPDH signal expressed relative to the no DIVAC control, which was set to 1. (D) Enhancer function of DIVAC sequences assayed in DT40 cells. Each DIVAC sequence was inserted downstream of a minimal promoter–luciferase gene cassette. Luciferase activity, corrected for transfection efficiency within individual experiments, was normalized to the activity found with the construct containing hIgλE, which was arbitrarily set to 1. Data are presented as the mean (± standard error of the mean) of 3–5 independent experiments. Two-tailed unpaired t-tests were used to compare data for the mIgλ enhancers and shadow enhancers. *p<0.05; **p<0.01.
Given the relatively strong DIVAC function associated with the mIgλ shadow enhancers, we wondered whether they also possessed transcriptional enhancer activity. To test this, sequences were cloned downstream of a minimal promoter–luciferase reporter and transfected into the UNG−/−AIDR recipient cell line used for the GFP4 studies. Both mIgλE3-1s and mIgλE2-4s were able to stimulate luciferase expression above that of the empty vector (no DIVAC) control, but both exhibited significantly less enhancer activity than their canonical mIgλ enhancer counterparts (Figure 7D), despite being stronger DIVAC elements. This discordance between transcriptional enhancer activity and DIVAC function further supports the conclusion that DIVAC operates by a mechanism distinct from that of stimulating transcription. A very recent study, published while our manuscript was under revision, identified the two mIgλ shadow enhancers based on epigenetic criteria and demonstrated that they possess B lineage–specific enhancer activity .
Using a highly sensitive, well-controlled assay we provide conclusive evidence that SH is targeted by Ig enhancer and Ig enhancer-like sequences. The phenomenon is strikingly conserved during vertebrate evolution, as even short mammalian Igλ and IgH enhancer fragments raised mutation rates more than 20-fold in chicken cells. SH activating sequences, or DIVAC, not only physically overlap the Ig enhancers but also closely resemble transcriptional enhancers in their mode of action by (i) requiring multiple transcription factor binding sites, (ii) functioning independent of orientation and when positioned either upstream or downstream of the transcription unit, and (iii) increasing activity through the collaboration of multiple enhancer-like regions, each of which depends on transcription factor binding motifs.
The recognition of Ig enhancers as SH targeting sequences yields a conceptual framework within which to reevaluate earlier studies. Most notably, the new results vindicate the early transgenic experiments that showed overlap of SH stimulating sequences with the Igλ, IgH intron, and Igκ enhancers , and synergistic effects between the Igκ intron and Igκ 3′ enhancer sequences ,. The failure of either Igκ intron or 3′ enhancer knockouts in mice to abrogate hypermutation , is consistent with the contributions of multiple, partially redundant Igκ enhancers to DIVAC function. Similarly, the failure of a previous study to identify SH targeting function associated with the Igκ intron and 3′ enhancers in DT40 cells  was likely due to use of a less sensitive assay and the absence of the Igκ distal enhancer. In addition, evidence that E-box ,,, NFκB , MEF2 , and PU.1-IRF4 , binding sites play a role in the targeting of SH or GCV can be explained by the importance of these sites within the context of Ig enhancers and enhancer-like sequences.
The results presented here provide the foundation for models of the cis-acting regulatory regions that target SH to a variety of Ig loci. The chicken Igλ locus is best understood and offers several lessons that might be generally applicable. In cIgλ, the enhancer cooperates with an evolutionarily conserved downstream element (3′Core) that itself possesses low levels of transcriptional enhancer activity (Figure 7D) but contains functionally important transcription factor binding motifs well known from Ig enhancers (Figures S2 and S3). However, it is clear that these two elements depend on additional sequences (e.g., Con2 and the region between cIgλE and 3′Core) for full DIVAC function (Figures 1 and S6) ,. The mouse Igλ and human and mouse Igκ loci offer parallels, with DIVAC function involving the combined action of two or more well-separated enhancer or enhancer-like elements. By analogy with cIgλ, it is tempting to think that other surrounding sequences further contribute to the full SH targeting activity of mammalian Ig loci. The human Igλ enhancer, the human and mouse IgH intron enhancers, and a combination of the known Igκ enhancers increase SH 20- to 30-fold in our assays, well below the 100-fold stimulation achieved by the full cIgλ DIVAC (Figure S6C). Indeed, previous analyses showing that deletion of mIgHEi or hIgHEi from the endogenous loci did not abolish SH , are consistent with the existence of other compensatory targeting elements, a strong candidate for which is the large 3′ regulatory region more than 200 kb downstream of IgHEi ,.
The identities of the trans-acting factors that bind Ig enhancers to stimulate SH are not known, although some candidates have been identified in previous studies and others can be inferred from the binding motifs whose integrity we show is important for DIVAC function. Substantial data support a role for E-box binding factors, including the E2a-encoded proteins E12 and E47 . Disruption of E2a in DT40 cells reduced the frequency of SH/GCV , as did overexpression of the E protein inhibitors Id1 and Id3 . E12 and E47 prefer to bind the CASSTG (S = C or G) subtype of E-box , and while mutation of this subtype reduces DIVAC function, mutation of E-boxes predicted to be bound poorly by E12/E47 does also . Existing data leave unresolved the identity of the E-box binding factor(s) that contribute to DIVAC function. Studies in DT40 have also implicated NFκB, PU.1, and IRF4 as trans factors relevant for the targeting of SH/GCV ,. Despite the fact that transcription and hypermutation enhancers make use of overlapping binding motifs and likely an overlapping set of trans factors, our data provide a compelling argument that the two processes operate by distinct mechanisms and, in particular, that DIVAC does not operate by increasing transcription.
It may not be a coincidence that enhancers, able to exquisitely regulate cell type– and gene-specific expression, have assumed the vital role of targeting SH to the Ig loci. The complex structure of DIVACs—distinct configurations of a common set of transcription factor binding motifs, with robust activity relying on multiple, and to some extent redundant, sequences—may reflect the formidable task of fine tuning and restricting SH. It might also reflect piecemeal evolution of DIVAC, with each Ig locus cobbling together an idiosyncratic collection of SH targeting elements. Chromosomal translocations near DIVACs likely increase the mutation rate in the neighborhood of the translocation breakpoint, as confirmed for the case of IgH to c-Myc locus translocations . It is also possible that non-Ig genes like BCL6 that mutate at substantial rates in AID-expressing B cells – do so because of DIVAC-like sequences in their neighborhoods. In support of this, a recent computational analysis found that promoter-proximal E-box, C/EBPβ, and YY1 binding motifs (all of which are found in some of the DIVAC elements identified here) were predictive of off-target SH of non-Ig genes .
Little is known about how gene-specific enhancers and particularly Ig enhancers distinguish themselves from other enhancers that may contain the same or similar transcription factor binding sites. Despite this limitation in our understanding of enhancer function, plausible models for how SH is targeted to Ig genes can be formulated based on what is known about the interaction of enhancers with the transcription initiation complex (Figure S7). One possibility is that a DIVAC-bound factor or a combination of factors actively recruit AID. A not mutually exclusive alternative is that DIVACs induce changes in the Pol II transcription initiation or elongation complex, making the transcribed DNA more accessible to AID. This hypothesis might explain why the accumulation of SH events rises rapidly downstream of the transcription start site and then falls off exponentially ,, and might establish a connection between DIVACs and stalled transcription ,,, or RNA exosome complexes . Interestingly, members of the APOBEC family can induce showers of clustered mutations in breast cancer and yeast cells that are believed to be related to single stranded DNA in the neighborhood of DNA double strand breaks ,, setting a precedent for how a change in DNA conformation can target deaminases to particular regions of the genome.
Materials And Methods
The GFP4 cassette (Figure 1A)—which resembles GFP2  but contains a 5′ untranslated sequence, the hypermutation target sequence (Figure S1), and, for increased GFP brightness, the GFPnovo2 open reading frame —was custom synthesized (Blue Heron Biotechnology) and cloned into the BamHI site of an AICDA locus–targeting construct , yielding the GFP4-containing, AICDA locus–targeting construct pAICDA_GFP4. A variant of pAICDA_GFP4, named pAICDA_GFP4D, was made in which the SpeI/NheI sites upstream of GFP4 were deleted and a unique NheI site was introduced downstream of GFP4. The cloning of DIVAC sequences into the GFP4 or GFP2 targeting vectors is described in Protocol S1.
Generation of the Recipient UNG−/−AIDR/puro Cell Clone
An UNG-deficient DT40 clone with both endogenous AICDA alleles deleted  was reconstituted with AID by the targeted integration of a bicistronic AICDA/gpt expression cassette into one of the AICDA loci . The second AICDA locus was subsequently marked by the targeted integration of a puromycin resistance gene driven by the chicken β-actin promoter, yielding the recipient UNG−/−AIDR/puro cell clone for transfections of GFP4 targeting constructs. The ΨV−IgL− clone in which the rearranged Igλ locus was replaced by a puromycin resistance cassette  was used for transfections of GFP2 targeting constructs.
Cell Culture and Flow Cytometry
DT40 cell culture, transfection, drug selection, and the identification of transfectants with targeted integration of GFP2 constructs were performed as described previously . Transfectants with targeted integration of GFP4 constructs were also detected by the appearance of puromycin sensitivity. The AID-negative UNG−/−AID−/−cIgλE↔3′Core clone was derived from the cIgλE↔3′Core transfectant by cre recombinase–mediated removal of the LoxP-flanked AICDA/gpt expression cassette .
GFP expression from GFP2 transfectants was assessed by flow cytometry at day 14 after subcloning, as described previously ,, whereas GFP4 transfectants were assessed at day 12 after subcloning. Details of the flow cytometry analysis are provided in Protocol S1.
Hypermutation Hotspot Sequence Analysis
Genomic DNA was isolated from a subclone of cIgλE↔3′Core after 6 wk of culture and used for the amplification of GFP4 sequences by PCR using Phusion polymerase (New England Biolabs). The PCR fragments were cloned using the In-Fusion Cloning Kit (Clontech) into the linearized pUC19 provided with the kit and sequenced. Thirty-four sequences covering the first 500 transcribed bases of GFP4 were aligned to the GFP4 sequence to detect sequence variation (Figure S1).
Orthologues of the Igλ locus were identified in the turkey, zebra finch, and ground finch genomes using the W fragment of cIgλ in low stringency blastn BLAST of the reference genome database and Blat genome searches of the respective genome sequences. BLAST and Blat searches were also used to identify the murine Igλ shadow enhancers and map them within the murine Igλ locus. The bird Igλ orthologues were aligned using the ClustalW2 web interface (http://www.ebi.ac.uk/Tools/msa/clustalw2/) to detect sequence contigs conserved during avian evolution. ClustalW2 was also used to create the other sequence alignments shown in Figures S2, S4, and S5. Searches for conserved transcription factor binding sites were performed using the TESS (Transcription Element Search Software) program .
Gene Expression Analyses
Reverse transcription quantitative PCR analysis was carried out on transfectants containing various DIVAC-GFP4 constructs after the cells were treated with 4-OH tamoxifen and subcloned to delete the AID expression cassette. This avoided potential effects on transcript levels due to nonsense-mediated mRNA decay. The resulting AID-negative cells used for analysis were stably GFP-positive (data not shown). RNA was extracted from 5×106 cells using the RNeasy Mini kit (Qiagen), and the cDNA was prepared from 1 µg of RNA using the iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using the DyNAmo HS SYBR Green qPCR kit (Thermo Scientific). GFP transcript levels were normalized to 18S rRNA levels. Samples were denatured for 15 min at 95°C, followed by 40 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. The primers used were as follows: GFPup-F 5′-ggaatatactttgccaagaagcgtt-3′, GFP5up-R 5′-accatcgttgccagaaccatt-3′, GFPcds-F 5′-gagcaaagaccccaacgaga-3′, GFPcds-R 5′-gtccatgccgagagtgatcc-3′, 18S-F 5′-taaaggaattgacggaaggg-3′, and 18S-R 5′-tgtcaatcctgtccgtgtc-3′.
RNA for Northern blot analysis was prepared from GFP2 or GFP4 cell lines with the RNeasy kit (Qiagen) or TRIzol reagent (Invitogen). 10 µg of total RNA was run on a gel, transferred to a membrane, and hybridized with a GFP probe. The blot was then stripped and reprobed with a GAPDH probe as a loading control. Using Image Lab software (Bio-Rad), bands were quantitated and normalized to the corresponding GAPDH signal, and values were presented relative to the GFP4 no DIVAC control. The probes were PCR-amplified DNA products made with the corresponding primers: GFPp-F 5′-accatggtgagcaagggcga-3′, GFPp-R 5′-ctaggacttgtacagctcgtccatgc-3′; GAPDHp-F 5′-accagggctgccgtcctctc-3′, GAPDHp-R 5′-ttctccatggtggtgaagac-3′.
Test sequences were cloned between SalI and BamHI sites downstream of the firefly Luc2 gene of the minimal promoter containing pGL4.23 vector (Promega). 20 µg of the plasmid was co-transfected into UNG−/−AIDR/puro cells with 2.5–5.0 µg of pGL4.75 Renilla luciferase control vector (Promega) using the Amaxa Nucleofector kit V (Nucleofector program B-023) (Lonza). The relative activity of firefly luciferase to Renilla luciferase was determined using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol.
Introduction of in-frame stop codons by transition mutations within the hypermutation target sequence of GFP4. The top line shows the first 500 base pairs downstream of the GFP4 transcription start site. The hypermutation target sequence starts with the underlined ATG start codon and ends with the Linker sequence followed by a XbaI site and the GFP open reading frame. Hypermutation hotspots (WRCY and its complement RGYW; W = A or T, R = A or G, Y = C or T) are shown in red, with the preferentially mutated base in bold. Mutations in 34 sequences from an UNG-deficient cIgλE↔3′Core subclone after 6 wk of culture are aligned below the GFP4 sequence, with mutations leading to stop codons in bold. When more than six mutations were seen at a given position, the total number is indicated with a subscript. One 3-bp deletion and a single transversion mutation are shown in blue.
Alignment of Igλ locus sequences from chicken, turkey, zebra finch, and ground finch. Conserved transcription factor binding motifs, identified as described in Materials and Methods, are indicated. Bases fitting the consensus of the binding motifs are in bold. (A) Con2 sequences containing a conserved E-box and two putative (p) IRF sites, referred to as pIRF-up and pIRF-down to distinguish the upstream and downstream sites. (B) cIgλE sequences containing conserved E-box, NFκB, MEF2, and PU.1-IRF4 binding motifs. (C) 3′Core sequences containing conserved E-box and putative core binding factor (CBF), C/EBP, and PU.1 binding motifs.
Deletion and mutation analysis of 3′Core, the second autonomous chicken Igλ DIVAC element. (A) Diagram of the chicken 3′Core fragment with truncations and deletions indicated below and conserved transcription factor binding motifs depicted as rectangles. The sequences of binding motifs and binding motif mutants are shown on the right. (B) GFP loss of subclones in the presence of full-length, truncated, and internally deleted 3′Core sequences. GFP4 assay. (C) Diagram of the chicken 3′Core fragment with binding motif deletions indicated below and conserved transcription factor binding motifs depicted as rectangles. The sequences of binding sites and of binding site mutants are shown on the right. (D) GFP loss of subclones in the presence of binding motif–deleted or binding motif–mutated 3′Core sequences. The first sample in (B) and (D) depict the same data as one another and as the 3′Core data of Figure 1D. GFP4 assay.
Alignment of vertebrate Igλ enhancer and mammalian IgHEi enhancer sequences. Conserved transcription factor binding motifs, identified as described in Protocol S1, are indicated. Bases fitting the consensus of the binding motifs are in bold. (A) The upstream and downstream E-box, as well as NFκB, MEF2, and PU.1-IRF4 binding sites are highlighted in the alignment of human, murine and chicken Igλ enhancer sequences. (B) Human and murine IgHEi enhancer sequences containing conserved YY1 (μE1), E-box (μE2 and μE4), Ets1 (μA), PU.1 (μB), IRF, and Octamer transcription factor binding sites. Also indicated are the well-studied μE5 and μE3 motifs ,,, which are not conserved at the sequence level between human and mouse. The mouse μE5 motif binds E proteins such as E47 . Despite poor conservation, the μE3 sites of both mouse and human have been suggested to bind the same factor (core binding factor, CBF) .
Alignment of the human and murine Igκ enhancer sequences. Conserved transcription factor binding motifs, identified as described in Materials and Methods, are indicated. Bases fitting the consensus of the binding motifs are in bold. (A) IgκEi sequences containing five conserved E-boxes (κE1, κE2, κE3, and two additional E-boxes in which the CANNTG motif is conserved) and a conserved NFκB binding site. (B) IgκE3′ sequences containing conserved E-box, NFκB, and PU.1-IRF4 binding sites. (C) IgκEd sequences containing conserved E-box, and putative NFκB, PU.1, and IRF binding sites.
Congruence between the GFP2 and GFP4 assays and analysis of synergy between cIgλE and Con2. (A) Diagram of the Con2-cIgλE region with truncations of Con2 indicated below and conserved transcription factor binding motifs depicted as rectangles. The sequences of binding motifs and binding motif mutants are shown on the right. (B) Flow cytometry profiles of representative subclones of primary transfectants carrying either GFP2 alone (AIDR) or combined with the cIgλ sequence specified above each plot, named according to (A) and Figure 1B. The transfectant named W carries the full-length cIgλ DIVAC sequence . All transfectants are UNG-proficient, AID-reconstituted. (C) GFP loss in the presence of the indicated DNA elements (GFP2 assay). (D) GFP loss in the presence of the indicated individual DNA elements or composite Con2-cIgλE elements containing full-length, truncated, or mutated Con2 sequences (GFP4 assay). The data for Con2-cIgλE are the same as in Figure 1D.
Model for the targeting of SH by Ig enhancers. Recruitment of lymphoid and general transcription factors (some candidate factors are shown [colored ovals]) to multiple Ig enhancer and enhancer-like sequences (blue ovals) (top). This leads to the formation of Ig enhancer–bound protein complexes that interact by looping with the transcription initiation complex assembled at the Ig promoter (middle). It is possible that Ig enhancer–bound protein complexes directly or indirectly recruit AID (purple oval) to the transcription initiation complex (middle) to facilitate SH of the Ig gene. Alternatively, or in addition, the transcription factors recruited by the Ig enhancers might alter parameters of transcription elongation (perhaps increasing Pol II pausing/stalling), thereby increasing the amount of single stranded DNA available for deamination by AID (bottom). While looping involving the enhancers is not depicted in this latter case, it could be occurring at the time of Pol II pausing/stalling.
Protocols used for target vector construction and FACS analysis. Detailed description of the methods used for the construction of the GFP4 and GFP2 targeting vectors and for the flow cytometry analysis of GFP fluorescence and calculation of GFP loss.
We are grateful to Abigail Raynor for deriving the sequences of mutated GFP4, and to Ranjan Sen, Filip Senigl, and Rashu Seth for helpful comments on the manuscript. This manuscript is dedicated to the memory of Michael Neuberger.
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: J-MB JA HA JJM DGS. Performed the experiments: J-MB JA HA JJM. Analyzed the data: J-MB JA JJM DGS. Wrote the paper: J-MB JA JJM DGS.
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