The contested role of uracil DNA glycosylase in immunoglobulin gene diversification

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15 Dauphinot, L. et al. (2005) The cerebellar transcriptome during postnatal development of the Ts1Cje mouse, a segmental trisomy model for Down syndrome. Hum. Mol. Genet. 14, 373–384 16 Daniel, A. et al. (2001) Karyotype, phenotype and parental origin in 19 cases of triploidy. Prenat. Diagn. 21, 1034–1048 17 Eakin, G.S. and Behringer, R.R. (2003) Tetraploid development in the mouse. Dev. Dyn. 228, 751–766 18 Hassold, T. et al. (1980) A cytogenetic study of 1000 spontaneous abortions. Ann. Hum. Genet. 44, 151–178 19 Hackshaw, A.K. and Wald, N.J. (2000) Revised distribution parameters for serum markers for trisomy 18. J. Med. Screen. 7, 215 20 Aitken, D.A. et al. (1993) First-trimester biochemical screening for fetal chromosome abnormalities and neural tube defects. Prenat. Diagn. 13, 681–689

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The contested role of uracil DNA glycosylase in immunoglobulin gene diversification Simonne Longerich and Ursula Storb Department of Molecular Genetics and Cell Biology, Committee on Immunology, University of Chicago, Chicago, IL 60637, USA

Class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes are initiated by the activation-induced cytosine deaminase AID. The resulting uracils in Ig genes were believed to be removed by the uracil glycosylase (UNG) and the resulting abasic sites treated in an error-prone fashion, creating breaks in the Ig switch regions and mutations in the variable regions. A recent report suggests that UNG does not act as a glycosylase in CSR and SHM but rather has unknown activity subsequent to DNA breaks that were created by other mechanisms.

Introduction B lymphocytes are unique in their specialization for antibody production. Functional antibody-encoding immunoglobulin variable (V) genes are rearranged from non-functional V, diversity (D) and joining (J) gene segments only in B cells. These cells can then further modify their V genes by somatic hypermutation (SHM), whereby mutations (mainly point mutations) accumulate in as much as 20% of the 1–2 kb DNA comprising the V gene and flanking regions, enabling antibodies with improved affinity to be selected for [1]. Immune effector mechanisms of antibodies are also improved by altering the constant (C) domain. Mature naı¨ve B cells express the Cm (and/or Cd) constant domain. In activated B cells, class switch recombination (CSR) eliminates Cm (and other C genes) in favor of a downstream C domain, for example, Cg1 (Figure 1), which encodes a C domain more appropriate for eliminating the activating antigen. CSR and SHM are clearly different types of genetic changes; Corresponding author: Storb, U. ([email protected]). Available online 5 March 2005 www.sciencedirect.com

however, both processes depend on the deamination of cytosine (C) to uracil (U) by the activation-induced deaminase, AID [1]. Uracil in DNA is normally removed by uracil glycosylases to leave an abasic site that is faithfully repaired. However, during CSR and SHM of Ig genes, U (and potentially neighboring bases) is subjected to error-prone mechanisms that are not fully understood (Figure 1). The finding that CSR is severely reduced and that SHM is altered in humans and mice that are deficient in the uracil DNA glycosylase (UNG) [2,3]: (i) lends support to the idea that the substrate of AID is DNA; (ii) demonstrates that UNG is the major DNA glycosylase in CSR and SHM; and (iii) suggests a model in which the abasic site is processed in an error-prone fashion to result in CSR and SHM [1]. A recent report from Begum et al. challenges this hypothesis [4]. Inactivation of UNG by Ugi CSR requires double-strand breaks (DSBs) in the participating Ig switch (S) regions (e.g. in Sm and Sg1 in cells that switch from IgM to IgG1). The DSBs are postulated to result from single-strand breaks on opposite strands in each of the participating S regions and were created by incisions that occurred subsequent to the removal of the AID-created uracil. To study the role of UNG in CSR, Begum et al. [4] expressed uracil DNA glycosylase inhibitor protein (Ugi) in a B-cell line, and found that CSR was reduced by w14-times when the cells were activated to perform CSR. This finding is consistent with the reduction of CSR seen in UNGK/K mice [3]. Unexpectedly, however, chromatin immunoprecipitation (ChIP) analysis showed that phosphorylated histones gH2AX were associated with DNA in the Sm region. H2AX has been shown to become phosphorylated at DSBs [5]. Moreover, gH2AX foci

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Figure 1. Models of somatic hypermutation (SHM) and class switch recombination (CSR). SHM results in the accumulation of point mutations (red asterisks) in DNA encoding the variable region of an antibody (VDJ region) (left panel). Activation-induced deaminase (AID, shown in red) is required for this process. With help from single-stranded binding protein (RPA, shown in purple), AID is thought to deaminate cytidines to create uracils (U) on either DNA strand in transcribed double-stranded DNA (horizontal arrow under VDJ–Cm represents the antibody-encoding transcript), mainly in the VDJ portion. Uracils can either be replicated to produce CG to TA transition mutations, or be removed from the DNA backbone by uracil glycosylase (UNG, shown in green) to result in an abasic site (gap). The three possible fates of the abasic site are: (i) error-free base-excision repair to restore the original CG basepair; (ii) replication using the abasic site as a template to result in any possible mutation (N); or (iii) nicking at the abasic site [e.g. by AP endonuclease (APE1), lilac trapezoid], removal of the abasic sugar-phosphate group and subsequent gap-filling by DNA synthesis with error-prone polymerases to produce one or more mutations. CSR results in joining of the variable region to a different constant region class (Cg3 through Ca, as arranged in mouse DNA, blue boxes along the top of the figure). Model 1 assumes similar functions of AID and UNG to those in SHM, except that double-strand breaks are created by simultaneous AID deamination, UNG uracil removal and DNA nicking nearby on cDNA strands in transcription-activated switch regions (Sm and Sg1). S regions are represented in orange and contain repetitive DNA sequences composed partly of AGCT tetramers (hotspots for AID targeting [1]), which precede each C region, except Cd. Further processing and joining of broken S regions is accomplished by unknown factors (depicted in blue) and can include the mismatch-repair protein, MSH2 (yellow). Model 2 is based on the report by Begum et al. [4], which stipulates that UNG glycosylase activity is not required for CSR. They hypothesize that cytosine(s) in an unknown mRNA might be deaminated by AID. The edited mRNA would then encode an endonuclease that creates DNA breaks in S regions. The role of UNG in this model is to recruit the unknown processing or joining factors that complete switch recombination. The normal products of switch recombination (hybrid Sm–Sg1 fusing the new C region to the variable region and the excised circle) are also shown.

have been observed in B cells induced to switch, and switching is impaired in H2AX-deficient mice [6]. Thus, ChIP of Sm DNA in Ugi-expressing cells suggests that AIDdependent DSBs in S-region DNA are created despite UNG deficiency. Why the cell-line data disagree with results from CSR-deficient patients with mutations in UNG, where DSBs (detected by ligation-mediated PCR) have not been observed [2], is unclear. Nevertheless, Begum et al. [4] conclude that AID-induced DSBs are independent of UNG activity and suggest that UNG is ‘required not for Sm cleavage, but probably for subsequent repair steps during CSR’. Honjo and collaborators favor the idea that breaks are caused by an unknown endonuclease, whose mRNA has been edited by AID [4,7] (Figure 1, model 2). Regarding the role of UNG, however, the authors fail to explain why it would be present at S regions in the absence of AID-induced uracils in S-region DNA. www.sciencedirect.com

Alternatively, gH2AX might not be a sure indicator of S-region DSBs. H2AX can become phosphorylated after treatment of cells with reagents that are not known to induce DSBs, such as hydroxyurea (J. Borowiec, personal communication) [8]. H2AX is phosphorylated by the ATR kinase [8,9], which is recruited to the foci of DNA repair, requiring the single-stranded DNA-binding protein, RPA [10]. RPA is also required for AID to deaminate cytosines on transcribed double-stranded DNA in vitro [11] (Figure 1). Therefore, Sm-associated gH2AX might mark the locations of AID deamination, rather than DSBs. Function of UNG mutants To further investigate the role of UNG, Begum et al. introduced mutant UNG into activated B cells from UngK/K mice [4]. Three variants, each with one

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point mutation that reduces UNG activity in vitro by 200–2500 times [12], enabled essentially normal levels of CSR to occur in activated B cells from UNGK/K mice. Begum et al. concluded that the reduction of CSR in UNGK/K mice is not due to the lack of uracil removal, but a result of a loss of an unknown activity of UNG that is retained in the UNG mutant proteins. Because all three single mutants retain DNA-binding activity [4,12], Begum et al. suggested that UNG might function as a scaffold in CSR. Alternatively, it is possible that UNG is not limiting in CSR, rather, the formation of uracil might be limiting. Because double-mutants of UNG transgenes did not rescue the UNG deletion, the greatly reduced but not completely abolished UNG activity of the single mutants might be sufficient to remove enough uracils to create abasic sites and induce base-excision repair (BER) leading to single-strand breaks and subsequent CSR [1]. DSBs in Ig switch regions in the absence of the uracil glycosylase activity of UNG How do breaks occur in S regions during CSR without the uracil glycosylase activity of UNG? There are several other uracil glycosylases that can compensate, for example, single-strand selective monofunctional uracil DNA glycosylase (SMUG1) [13]. Its activity is low in lymphoid cells, and the ability of UNG to remove uracil seems to be an order of magnitude greater than SMUG1 [14]. Over-expression of SMUG1 does not rescue the CSR deficiency in UNGK/K B cells [14], but its ability to initiate BER and result in DSBs was not tested. Similarly, thymine DNA glycosylases, methyl-CpG binding domain protein 4 (MBD4) and endonuclease VIII-like 1 (NEIL1) can remove uracil and therefore result in DNA breaks, although inactivation of MBD4 does not reduce CSR or SHM [15]. According to Begum et al. [4], the processing and joining of the broken double-strand ends in the donorand acceptor-switch regions would then require UNG or catalytically inactive UNG to act as a scaffold (Figure 1, model 2). UNGK/K mice show greatly reduced CSR, but not its complete elimination [3,4]. However, in mice with inactivation of both UNG and mutS homolog 2 (MSH2), CSR is essentially eliminated, at least in vivo [14]. This suggests that mismatch repair (MMR) might represent a second pathway to create DNA breaks from U:G mismatches in the S regions. Inactivation of MSH2 alone has a major effect on CSR [16], whereas inactivation of mutS homolog 6 (MSH6) reduces CSR to 30–60% of wild-type levels [17]. Thus, MSH2 might have more than one role in CSR. It forms hetero-dimers with Msh6, which act in MMR of singlebase mismatches, and hetero-dimers with Msh3 for MMR of single-base insertions and/or deletions and larger mismatches [18]. The patterns of switch junctions are normal in Msh6K/K mice but are altered in Msh2K/K mice [17]. Li et al. suggest that Msh2 in cooperation with Msh3, but not Msh6, is required for the ‘processing [of] non-homologous ends created by staggered DNA breaks’ [17]. Studying CSR in UngK/K;Msh6K/K mice might reveal whether MSH2 and MSH6 recognition of www.sciencedirect.com

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AID-dependent U:G mismatch has a role in CSR, when MSH2 and MSH3 can still function to remove singlestranded overhangs [19]. UNG in SHM AID-dependent SHM affects the S-region sequences and the V regions of activated B lymphocytes [6]. Begum et al. found mutations in the 5 0 Sm region of the activated Ugi-expressing B-cell line at the same frequency as in Ugi-negative cells. This is consistent with the findings in UNGK/K mice where the mutation frequency is unaffected [3]. However, in UNGK/K cell lines, w100% of the mutations were transitions from C or G, with and without Ugi [4], suggesting that CG to TA transitions were created by direct replication of uracil. In the 5 0 Sm region of wild-type mice, only 41% of the mutations from C or G were transitions, the remainder also including mutations from A or T [6]. It will, therefore, be important to investigate SHM in Ig variable regions of UNGK/K mice that express the UNG-mutant transgenes used by Begum et al. [4] to determine if ‘catalytically inactive’ UNG can rescue the SHM pattern. If the UNG mutants rescue CSR but not the SHM pattern, one would conclude that the catalytic function of UNG is required for SHM, and that DSBs can be created in CSR by other mechanisms (e.g. MMR) but that UNG is required for processing downstream of the DSBs. Concluding remarks The currently available data [2,4,14] do not permit a distinction between a catalytic role of UNG (i.e. uracil removal) or a scaffold or other unknown role in CSR and SHM. It needs to be determined directly whether DSBs can still occur in the absence of UNG and whether UNG with greatly reduced catalytic activity can rescue CSR but not SHM in UNGK/K mice. Acknowledgements S.L. was supported by an international fellowship from the AAUW Educational Foundation. S.L. and U.S. are supported by NIH grants AI47380 and AI53130. We thank T.E. Martin for critical reading of the article.

References 1 Storb, U. and Stavnezer, J. (2002) Immunoglobulin genes: generating diversity with AID and UNG. Curr. Biol. 12, R725–R727 2 Imai, K. et al. (2003) Human uracil–DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat. Immunol. 4, 1023–1028 3 Rada, C. et al. (2002) Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 4 Begum, N.A. et al. (2004) Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch. Science 305, 1160–1163 5 Redon, C. et al. (2002) Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12, 162–169 6 Petersen, S. et al. (2001) AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665 7 Begum, N.A. et al. (2004) De novo protein synthesis is required for activationinduced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. U. S. A. 101, 13003–13007

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8 Ward, I.M. and Chen, J. (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276, 47759–47762 9 Fernandez-Capetillo, O. et al. (2004) H2AX: the histone guardian of the genome. DNA Repair (Amst.) 3, 959–967 10 Zou, L. and Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 11 Chaudhuri, J. et al. (2004) Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998 12 Mol, C.D. et al. (1995) Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell 80, 869–878 13 Nilsen, H. et al. (2000) Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication. Mol. Cell 5, 1059–1065 14 Rada, C. et al. (2004) Mismatch recognition and uracil-excision provide complementary paths to both immunoglobulin switching and the second (dA:dT-focussed) phase of somatic mutation. Mol. Cell 16, 163–171

15 Bardwell, P.D. et al. (2003) The G-U mismatch glycosylase methyl-CpG binding domain 4 is dispensable for somatic hypermutation and class switch recombination. J. Immunol. 170, 1620–1624 16 Ehrenstein, M. and Neuberger, M. (1999) Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin classswitch recombination: parallels with somatic hypermutation. EMBO J. 18, 3484–3490 17 Li, Z. et al. (2004) Examination of Msh6- and Msh3-deficient mice in class switching reveals overlapping and distinct roles of MutS homologues in antibody diversification. J. Exp. Med. 200, 47–59 18 Kolodner, R.D. and Marsischky, G.T. (1999) Eukaryotic DNA mismatch repair. Curr. Opin. Genet. Dev. 9, 89–96 19 Sugawara, N. et al. (1997) Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proc. Natl. Acad. Sci. U. S. A. 94, 9214–9219

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Genome Analysis

Biased codon usage near intron-exon junctions: selection on splicing enhancers, splice-site recognition or something else? Jean-Vincent Chamary and Laurence D. Hurst Department of Biology and Biochemistry, University of Bath, Bath, UK, BA2 7AY

Two groups recently argued that, in human genes, synonymous sites near intron-exon junctions undergo selection for correct splicing. However, neither study controlled for the possibility of an underlying nucleotide bias at the ends of exons. In this article, we show that generalized A and T enrichment exists, which could be independent of splicing regulation. Evidence for selection between synonymous codons that are associated with splicing enhancers remains after controlling for this bias, whereas support for cryptic splice-site avoidance is diminished.

Introduction Although synonymous sites are usually thought to evolve neutrally in mammals (e.g. Refs [1,2]), recent evidence suggests otherwise [3–8]. In addition, two groups [9,10] have recently claimed that, near intron-exon junctions, selection for efficient intron excision influences synonymous codon choice. Willie and Majewski [10] reported that, in humans, GAA is more abundant near junctions than GAG, attributing this to the role of GAA as an exonic splicing enhancer (ESE, [11]). This we call the ‘enhancer model’. This model fits with evidence that a region of the breast cancer gene, BRCA1, with an unusually reduced synonymous substitution rate [12], is a splicing enhancer [13,14] and that, more Corresponding author: Hurst, L.D. ([email protected]). Available online 12 March 2005 www.sciencedirect.com

generally, there is a reduced rate of single nucleotide polymorphisms (SNPs) in ESEs [15]. However, results from Eskesen et al. [9] support a different set of hypotheses, which we call the ‘cryptic splice-site avoidance model’. They postulated that, because the 3 0 -ends of introns typically terminate AG, exons should avoid using this dinucleotide at the 5 0 -end to minimise the chance of deleterious aberrant splice forms. Disentangling cryptic splice-site avoidance from selection on splicing enhancers The interpretation of both sets of results is problematic. All of the effects described above have one thing in common: preference for A rather than G near intronexon junctions. To demonstrate that the suggested interpretations are correct, one must also show that enrichment of A is specific to codons that are associated with splicing, and not a general bias for A. Indeed, a decline in GC content approaching junctions was noted by Majewski’s group [10,16], although this might simply reflect GAA preference. Therefore, we first asked whether such a bias exists in codons that are not known to be associated with splicing. Because we found that generalized AT enrichment occurs, the remaining issue is whether we can still find evidence for the forces proposed by the two groups, when controlling for this bias. Our analysis also attempts to discriminate between the two models. According to the cryptic splice-site avoidance