A Role for DNA Polymerase {micro} in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joiningprocess that resolves recombination-activating gene (RAG)-induceddouble-strand breaks and results in V(D)J gene rearrangementsvary during mammalian ontogeny. In the mouse, the first immunoglobulingene rearrangements emerge during midgestation periods, buttheir repertoires have not been analyzed in detail. We decidedto study the postgastrulation DJ_H joints and compare them withthose present in later life. The embryo DJ_H joints differedfrom those observed in perinatal life by the presence of shortstretches of nontemplated (N) nucleotides. Whereas most adultN nucleotides are introduced by terminal deoxynucleotidyl transferase(TdT), the embryo N nucleotides were due to the activity ofthe homologous DNA polymerase µ (Polµ), which waswidely expressed in the early ontogeny, as shown by analysisof Polµ^–/– embryos. Based on its DNA-dependentpolymerization ability, which TdT lacks, Polµ also filledin small sequence gaps at the coding ends and contributed tothe ligation of highly processed ends, frequently found in theembryo, by pairing to internal microhomology sites. These findingsshow that Polµ participates in the repair of early-embryo,RAG-induced double-strand breaks and subsequently may contributeto preserve the genomic stability and cellular homeostasis oflymphohematopoietic precursors during development.

INTRODUCTION

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INTRODUCTION

The adaptive immune system is characterized by the great diversityof its antigen receptors, which result from the activities ofenzymatic complexes that cut and paste the genomic DNA of antigenreceptor loci. The nonhomologous end-joining (NHEJ) machineryis then recruited to repair the double-strand DNA breaks (DSBs)inflicted by the products of the recombination-activating genes(RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptorrepresents a singular shuffling of two heavy (H) and two light(L) chains, which are derived from the recombination of V, D,and J gene segments of the IgH locus and of V and J for IgL(71). Besides these combinatorial possibilities, most Ig variabilityderives from extensive processing of the coding ends, includingexonucleolytic trimming of DNA ends, together with the additionof palindromic (P) nucleotides templated by the adjacent germline sequence and of nontemplated (N) nucleotides secondaryto the activity of the terminal deoxynucleotidyl transferase(TdT), a lymphoid-specific member of family X of DNA polymerases(reviewed in reference 56). During B-lineage differentiation,IgH rearrangements occur before those of the IgL locus, andD-to-J_H rearrangements precede V-to-DJ_H rearrangements (62).DJ_H joints are formed in any of the three open reading frames(ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribedas a Dµ protein that provides negative signals to theB-cell precursors, and ORF3 frequently leads to stop codons(32, 33, 37). Germ line V, D, and J gene segments display shortstretches of mutually homologous nucleotides (SSH), which arefrequently used in gene rearrangements during perinatal periods,when N additions are absent (27, 32, 55, 57). The actual IgV-region repertoires represent both the results of the NHEJprocess associated with genomic VDJ recombination and thoseof antigen-independent and -dependent selection events. Althoughthe core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNAligase IV) are by themselves able to join RAG-induced, incompatibleDNA ends, family X DNA polymerases can be recruited to fillgaps created by imprecise coding ends with 3' overhangs (DNApolymerase µ [Polµ] and Pol ${lambda}$ ) and/or to promote diversitythrough the addition of N nucleotides (TdT) (34, 56).

The lymphoid differentiation pathways and clonotypic repertoiresare developmentally regulated and differ between the embryo-fetaland adult periods (2, 44, 68). The perinatal B cells resultfrom a wave of B lymphopoiesis occurring during the last thirdof mouse gestation (13, 14, 21, 70). Perinatal V_H gene usagediffers from that predominating in the adult (1, 69), and theformer VDJ joints rarely display N additions, leading to V-regionrepertoires enriched in multi- and self-reactive specificities(36, 40). The program of B-cell differentiation starts at embryonicdays 10 to 11 (E10 to E11) in embryo hematopoietic sites, afterthe emergence of multipotent progenitors (at E8.5 to E9.5) (18,19, 23, 31, 51, 73). DJ_H rearrangements were detected in theseearly embryos, whereas full VDJ_H sequences were not observedbefore E14 (14, 18, 51, 66), when VJ ${kappa}$ rearrangements were alsofound (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzedto date corresponded to late fetuses (E16) (14, 53). We reasonedthat the true baseline of the Ig rearrangement process occursin midgestation embryos, when the first DJ_Hs are not yet transcribedand, consequently, not subjected to selection and are conditionedonly for the evolutionarily established and developmentallyregulated usage of distinct NHEJ machineries.

We report here the sequence profiles of the earliest embryoE10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_Hjoints bearing N nucleotides, in the absence of detectable TdTexpression, were found. Moreover, the embryo DJ_H joints lackingN nucleotides (N^–) used fewer SSH to recombine than newbornDJ_Hs, and these SSH were widely dispersed along the embryo Dsequences, in contrast to the most joint-proximal ones, whichpredominated in newborn DJ_Hs. Considering that Polµ isthe closest relative of TdT (42% amino acid identity) (22),which is able to introduce N nucleotides in vitro (4, 22, 34,39, 49) and to join DNA ends with minimal or even null complementarity(17, 58), and that it is expressed in early-embryo organs, wedecided to investigate its putative contribution to the firstembryo DJ_H joints. The DJ_H joints obtained from Polµ^–/–embryos (48) showed a significant reduction of N nucleotidescompared to wild-type (WT) embryos. Moreover, highly preservedDJ_H joints (with <3 deleted nucleotides) were selectivelydepleted in the Polµ^–/– mouse embryos, whilethe remaining DJ_Hs preferentially relied upon longer stretchesof homology for end ligation. These findings support the ideathat Polµ is active during early-embryo DJ_H rearrangementsand that both its template-dependent and -independent ambivalentfunctions may be used to fill in small nucleotide gaps generatedafter asymmetric hairpin nicking and also to extend coding endsvia a limited TdT-like activity.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Embryo microsurgery and cell purifications. BALB/c, BALB/c.RAG-2^–/–, and BALB/c x 129/SV Polµ^–/–(nine generations backcrossed to BALB/c) mice were maintainedin the animal facilities of the Instituto de Salud Carlos III,Madrid, Spain, and the Centro de Biología Molecular SeveroOchoa, Madrid, Spain. The gestational age was determined bythe vaginal plug after overnight mating (E0). Eggs containingthe embryo attached to the placenta were washed by sequentialtransfers through 50-ml phosphate-buffered-saline tubes. Afterremoval of the placenta, the embryo proper and yolk sac wereseparated and washed out again by passages through 20-ml petridishes (51). All animal studies were performed in accordancewith Spanish animal protection laws. The cells were incubatedwith anti-CD19-phycoerythrin (1D3), anti-CD45R/B220-allophycocyanin(RA3-6B2), anti-CD45-allophycocyanin (30F11), anti-cKit-fluoresceinisothiocyanate (2B8), and anti-IgM-fluorescein isothiocyanate(331.13) antibodies (BD Pharmingen). Nonspecific backgroundwas eliminated by incubations with 10% mouse serum and Fc-Block(BD Pharmingen) and controlled with isotype-matched irrelevantantibodies. Cell debris and dead cells were discarded by adjustinglight parameters and by propidium iodide staining. Labeled cellswere purified in a DIVA cell sorter (BD Pharmingen), the degreeof purity was controlled in a FACSCalibur with Cellquest software(BD Pharmingen), and only samples more than 95% pure were processed.

RT-PCR, genomic PCR, cloning, and sequencing. Genomic DNA and total RNA were extracted, and oligo(dT)-primedcDNA samples were prepared with avian myeloblastosis virus reversetranscriptase (RT), as described previously (51). PCR amplificationswere performed with 1 U of AmpliTaq Gold DNA polymerase (RocheMolecular Systems) in a PTC-200 DNA Engine cycler (Bio-Rad).DJ_H rearrangements were detected with a genomic amplificationassay by using the Faststart PCR amplification kit (Roche MolecularSystems). DNA templates corresponding to 10³ cells were amplified.For embryo-derived DJ_H rearrangements, 1 µl of the firstPCR amplification reaction mixture was subjected to a nestedPCR for 20 additional cycles. The primers and PCR conditionswere as indicated in Table S1 in the supplemental material.In the case of newborn-derived samples and to facilitate cloningof the larger DJ_H structures, nested amplifications were performedby using JH intron-specific oligonucleotides as 3' primers.The products of the primary (for J_H4) or nested (J_H1 to -3)PCRs obtained from adult and newborn samples, containing differentDJ_H rearrangements in each of the J_H-specific bands of the samesize, were cloned using the pMBL-T vector kit (Dominion MBL)and transformed into JM109 Escherichia coli competent cells(Promega). After being plated, colonies were amplified, theirproducts were separated electrophoretically on 2% agarose gels,and the bands were visualized with ethidium bromide and cleanedor gel purified with either PCR clean-up kits or gel spin kits(MoBio Laboratories), respectively. The purified bands weresequenced in an ABI7000 automatic sequencer with the BigDyesequencing mixture (Applied Biosystems).

The relative expression levels of TdT, Polµ, Pol ${lambda}$ , andG ${alpha}$ S genes were calculated by real-time PCR, performed on theLightCycler 2.0 system, by using the LightCycler FastStart DNAMaster SYBR green I kit (Roche). The cycling steps were as follows:1 cycle of 95°C for 10 min, followed by 45 cycles of 95°Cfor 10 s, 60°C for 10 s, and 72°C for either 15 s (TdT,Polµ, and Pol ${lambda}$ ) or 30 s (G ${alpha}$ S), with a final cycle of 72°Cfor 5 min, before a melting curve was performed. LightCyclersoftware 4.0 was used to calculate the threshold cycle (C_T)of each reaction, and the relative amount of specific cDNA oneach sample was determined using the 2^–CT method (46)by using G ${alpha}$ S as the internal control gene. The values obtainedfor newborn samples were used as calibrators to determine thechanges in the specific gene expression levels.

In vitro terminal-transferase assay. Terminal-transferase-mediated incorporation of a single deoxynucleotide(ddTTP) was assayed as described previously (39) on double-strandedDNA molecules differing in the number of 3' protruding nucleotides.The strand providing the 3' overhang (5'-CGCAAGTCAGCGCTACGGG[T]_0-5)was 5' labeled with [ ${gamma}$ -³²P]ATP and T4 polynucleotide kinase andhybridized to the unlabeled complementary strand (5'-CCCGTAGCGCTGACTTGCG)in the presence of 50 mM Tris-HCl (pH 7.5) and 0.3 M NaCl. Thereaction mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol,1 mM MnCl₂, 5 nM of labeled DNA, 200 nM of either human Polµor TdT, 100 µM ddTTP, 4% glycerol, and 0.1 mg/ml bovineserum albumin in 12.5 µl. After incubation (37°C;30 min), reactions were stopped by adding gel-loading buffer(95% formamide, 10 mM EDTA, 0.1% [wt/vol] xylene cyanol, and0.1% [wt/vol] bromophenol blue). The +1 extension products wereanalyzed by 8 M urea-20% polyacrylamide gel electrophoresis,detected by autoradiography, and quantitated with a phosphorimager.

Statistical analyses. GraphPad Prism 3.0 software was used. Means, standard deviations,and standard errors of the means (SEM) were calculated, andthe normality of the distributions was determined with the Kolmogorov-Smirnovtest. Comparisons of means were performed with the two-tailedunpaired t test with the Welch correction, from which P valueswere derived. For nonparametric samples, one-way analysis ofvariance with the Kruskal-Wallis test was performed. Contingencytables were used to compare frequencies among experimental groups,using chi-square and Fisher's exact tests to calculate P values.Deletion profiles were obtained by Gaussian kernel density estimation(The R Project for Statistical Computing [http://www.r-project.org]).

Nucleotide sequence accession numbers. The sequences analyzed in this study are posted as supplementalmaterial (see Fig. S1 to S5 in the supplemental material) andhave been deposited in EMBL with the following accession numbers:FM162406 to FM162492 (WT embryos); AM998795 to AM998802, FM161938to FM161957, and FM161968 to FM161985 (newborns); FM161991 toFM162014 and FM162405 (adults); FM162395 to FM162397 and FM162493to FM162508 (RAG2^+/– embryos); and FM162509 to FM162556(Polµ^–/– embryos).

RESULTS

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RESULTS

The first DJ_H rearrangements of the mouse embryo. A genomic PCR simultaneously amplified DJ_H1 to -4 gene rearrangementsand revealed DJ_H-specific bands in hematopoietic organs of synchronizedE10 to E12 BALB/c mouse embryos, as described previously (51).Fifty-eight percent of the PCRs performed in samples derivedfrom a mean of 10 embryo explants per experiment were positive,and of these, two-thirds showed a unique DJ_H band, suggestingthat they represented the earliest-appearing rearrangementsduring postgastrulation periods (Table 1; see Fig. S1 in thesupplemental material). From E13 onward, the DJ_H rearrangementslogarithmically expanded in the liver (14, 51). Control DJ_Hgroups recovered from 1-day-old livers and spleens and from3-month-old spleens were included (see Fig. S2 and S3 in thesupplemental material). DFL16.1 was the most utilized D gene,although this preference was more pronounced among newborn DJ_Hs.All J_H genes were similarly used in embryo and newborn samples,but most adult sequences contained the smallest DJ_H4 rearrangement,which was more easily cloned when the four DJ_H rearrangementswere present in the PCR samples. Both adult and newborn DJ_Hspreferentially utilized ORF1, whereas E10 to E12 embryo DJ_Hsshowed roughly similar frequencies of the three ORFs, indicatingtheir unselected character.

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TABLE 1. Characteristics of DJ_H joint sequences in BALB/c mouse embryos, newborns, and adults

E10 to E12 DJ_H coding joints include N nucleotides. The processing of DJ_H joints is conditioned by at least fourmechanisms: (i) usage of SSH in the recombination of two codingends, (ii) exonucleolytic nibbling of germ line sequences, (iii)inclusion of templated P nucleotides, and (iv) random additionof N nucleotides by TdT. TdT is highly expressed in adult pro-Bcells and hardly or not at all expressed in perinatal lymphoidprogenitors (8, 44). In agreement with this, most of the adultspleen DJ_Hs (80%) and only 2 out of 57 newborn DJ_Hs (3.5%) showedN nucleotides. Unexpectedly, 51% of E10 to E12 DJ_Hs includedN nucleotides (47 out of 92; P < 0.001 and P < 0.01 withrespect to newborn and adult DJ_Hs, respectively) (Fig. 1, left).The lengths of embryo N stretches per N⁺ sequence were lessthan those of adult DJ_Hs (3.2 ± 0.3 and 4.6 ±0.7 for embryo and adult DJ_Hs, respectively; mean ± SEM;P = 0.051). Considering that each exsanguinated embryo (andits dissected organs) was sequentially diluted in large buffervolumes (well over 10⁶ times its own volume), the possibilityof surgical contamination with B cells from the adult mice waslow. Alternatively, DJ_H joints could represent mother-derivedchimerism, although only a transient wave of maternal macrophageshas been detected in E7.5 to E9.5 embryo yolk sacs and no othercell chimerism in fetuses up to E16 (6, 52). Additional supportfor the embryo origin of the N⁺ DJ_Hs came from their unbiasedORF usage, while ORF2 was absent among adult N⁺ DJ_Hs (P <0.01 compared to embryo ORF2 usage) (Fig. 1, right); the slightpreference for ORF1 in total embryo DJ_Hs (Table 1) was actuallyaccounted for by their N^– sequences (data not shown).However, to definitively discard a maternal origin of the N⁺DJ_Hs, we sequenced DJ_Hs from embryos derived from BALB/c.RAG2^–/–females mated to BALB/c males. They also displayed an unselecteddistribution of ORFs, and 35% of these DJ_Hs showed stretchesof N nucleotides (Fig. 1, left; see Fig. S4 in the supplementalmaterial), confirming that the E10 to E12 N⁺ DJ_H sequences wereof embryonic origin.

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FIG. 1. N nucleotides in DJ_H joints derived from E10 to E12 BALB/c embryos. On the left, the frequencies of N⁺ DJ_H joints in adults (black), newborns (gray), WT embryos (hatched), and RAG2^+/– embryos derived from BALB/c male x BALB/c.RAG2^–/– female matings (crosshatched) are shown. The distribution of ORFs (circled numbers) among N⁺ DJ_H joints of adults and embryo DJ_H joints is displayed on the right. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns, not significant.

Developmental shifts of SSH utilization, D recombination sites, and nucleotide deletions in DJ_H joints. It is thought that the SSH present in D and J_H gene segmentspromote recombination whenever TdT is absent and N nucleotidesare not introduced (28, 57). In agreement with this, 12% ofadult and up to 78% of newborn DJ_Hs used such microhomologies.In contrast, both total and N^– embryo DJ_Hs showed lowerusage of SSH-related recombination sites than the equivalentDJ_Hs of newborns (P < 0.001 and P < 0.01, respectively)(Fig. 2A). The analysis of D recombination sites also revealeddistinctive patterns in both embryo and newborn DJ_Hs. The mostjoint-proximal nucleotide was preferentially used to ligateboth embryo and newborn DJ_Hs, yet this recombination point wasmuch more frequent in newborn than in embryo DJ_Hs (30/57 and35/108 DJ_H joints, respectively, ligating at position 1 versustotal DJ_H joints [BALB/c and RAG^+/– embryos are consideredtogether hereafter]; P < 0.01). Compared to those of newborns,the embryos’ SSH-related recombination sites were distributedalong the whole D segment sequence. When these SSH were classifiedbetween joint-proximal (first four D nucleotides) and joint-distalgroups, the latter appeared more frequently in embryo than innewborn DJ_Hs (P < 0.01) (Fig. 2B, left). The coding-end sequencesmay influence the DJ_H joint-processing events (3, 24, 29, 43,74); however, the observed embryo/newborn differences appliedequally to DJ_Hs using the single DFL16.1 gene (P < 0.01)and to the DSP2 family members (P < 0.05) (Fig. 2B, right).The reduced numbers of DSP2 gene-specific newborn DJ_Hs precludeda gene-by-gene comparison with the equivalent embryo DJ_Hs. Theincreased utilization of joint-distal D recombination positionsin embryo DJ_Hs resulted in larger DJ_H nucleotide deletions:Both DFL16.1⁺ and DSP2⁺ newborn DJ_H coding ends were more "protected"than the equivalent embryo DJ_Hs (P < 0.01) (Fig. 2C). Weconclude that (i) embryo N^– DJ_H joints used lower frequenciesof SSH than newborn N^– DJ_Hs, (ii) embryo DJ_Hs frequentlyrearranged to joint-distal SSH of D segments, and (iii) embryoDJ_Hs supported larger nucleotide deletions than newborn DJ_Hs.

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FIG. 2. Developmental shifts of SSH usage, of D recombination sites, and of nucleotide deletions in DJ_H joints. (A) Frequencies of usage of SSH-related recombination sites in total and N^– embryo and newborn DJ_Hs. (B) Embryo and newborn DJ_H joints recombine at SSH⁺ D sites. The DJ_H joints are arbitrarily located over the most joint-proximal SSH nucleotide; the progressively darker shades correspond to DJ_H joints using SSH 1, 2, 3, and >3 nt long; joint-proximal DJ_Hs are delimited between the two vertical dotted lines (upper left). The two upper-right bar graphs represent the distributions of D recombination sites in DFL16.1⁺- and DSP2⁺-specific SSH⁺ DJ_Hs (the nucleotide sequences are shown on the vertical axes, and for DSP2 genes, the numbers indicate variable nucleotide positions among the various gene family members). The bars below show the frequencies of joint-proximal DJ_Hs in total, as well as in D family-specific, embryo and newborn SSH⁺ DJ_Hs. (C) Degrees (means plus SEM) of nucleotide deletion in DFL16.1- and DSP2-specific embryo, newborn, and adult DJ_H joints. The symbols are as in Fig. 1, except for the shading of the upper bars in panel B.

High expression of Polµ in the early mouse embryo. The expression of TdT, Polµ, and Ku80 genes in B-cellprogenitors during mouse ontogeny was analyzed by conventionalRT-PCRs in purified CD19⁺ IgM^– cells from adult bone marrow(BM) and newborn liver, together with cKit⁺ CD45⁺ cells fromembryo hematopoietic sites, which included a small subset ofCD19⁺ B-cell precursors (5%) (19). TdT was present in adultBM, expressed at highly reduced levels in newborn pre-B cells,and undetected in embryo cells (even when a nested RT-PCR assaywas used) (data not shown). In contrast, Polµ transcriptswere more conspicuous in embryo cKit⁺ CD45⁺ cells than in bothadult and newborn samples. The NHEJ core Ku gene was also wellexpressed in adult, embryo, and newborn samples (Fig. 3A). Tobetter quantify the relative expression of X family DNA polymerasegenes, we undertook real-time PCRs for TdT, Polµ, andPol ${lambda}$ genes in the above-mentioned populations of adult BM, newbornliver, and embryo liver cells. Compared to newborn values, theadult TdT expression was 50- to 100-fold increased, while itwas completely absent in the embryo samples. In contrast, Polµrelative transcript levels were 5- to 10-fold higher in theembryo than in the newborn and adult cell samples. Pol ${lambda}$ expressionwas also slightly increased in the embryo samples (Fig. 3B).

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FIG. 3. Differential expression of TdT, Polµ, Pol ${lambda}$ , and Ku80 in lymphohematopoietic progenitors during mouse ontogeny. (A) CD19⁺ IgM^– B-cell precursors were purified from adult BM and newborn liver, and cKit⁺ CD45⁺ cells were purified from the indicated embryo organs (YS, yolk sac; AGM, aorta-gonad-mesonephros region). Semiquantitative RT-PCRs were done as described in Materials and Methods. G ${alpha}$ S gene expression was used as a reference for the total mRNA per sample. The negative and positive control samples were non-cDNA and adult thymus, respectively. (B) Normalized TdT, Polµ, and Pol ${lambda}$ transcript levels relative to newborn 2^–CT. The expression of each specific gene was normalized to that of the control G ${alpha}$ S gene, and the values obtained for both adult and embryo samples are relative to those obtained for newborn samples. The data shown are the means plus standard deviations of at least three different samples per experimental group. The symbols are as in Fig. 1.

Polµ and TdT polymerases show different size preferences for extending double-stranded DNA with 3' overhangs. The finding of N additions in the embryo DJ_Hs when TdT was absentbut Polµ was highly expressed prompted us to analyze thedifferential efficiencies of both family X polymerases at introducingN nucleotides in vitro into different double-stranded DNA substrates,as they might be found in vivo in V(D)J rearrangements. As seenin Fig. 4, blunt ends were inefficient substrates for both enzymes.TdT also could not extend short protruding ends (1 or 2 nucleotide[nt] residues) but was active on >2-nt-long 3' protrudingends. Polµ showed the highest efficiency on DNA ends withsmall 1- or 2-nt overhangs, later declining to its lowest levelat 4- or 5-nt overhangs, those in which the TdT activity wasmaximal. In absolute terms, the terminal-transferase activityof Polµ on its optimal substrates was about threefoldlower than that of TdT on ends protruding 4 nt. A mechanisticexplanation is that the formation of a precatalytic ternarycomplex for terminal-transferase activity is a rate-limitingprocess in the case of Polµ, which would thus avoid excessiveN additions and consequently favor templated insertions (P.Andrade, F. J. Lopez de Saro, and L. Blanco, unpublished data).

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FIG. 4. Differential in vitro terminal-transferase activities of Polµ and TdT. The assays were done on various double-stranded DNA substrates, either blunt ended or having increasing numbers of 3' protruding T residues. The percentage of extended DNA of each type was related to the length of the blunt-ended molecule and is represented in a combined histogram (black and gray bars for Polµ and TdT, respectively; means plus SEM; n = 3 independent experiments).

Polµ contributes to the processing of mouse embryo DJ_H joints. The lack of TdT, together with the potential of Polµ toadd N nucleotides in DSBs (see above) (34, 39, 58) and its involvementin V(D)J rearrangements (4, 5), prompted us to investigate itsputative activity in embryo DJ_H joints by analyzing Polµ^–/–embryo mice. Fewer and late-appearing DJ_H rearrangements weredetected in Polµ^–/– compared to WT embryos(25 and 67% of E11 DJ_H⁺ samples, respectively), suggesting thatthe V(D)J rearrangement process is less efficient in the mutantmice. Polµ^–/– embryo DJ_H joints were equallydistributed between DFL16.1- and DSP2-bearing sequences, utilizedall the JH gene segments, and showed an unbiased ORF distribution(see Fig. S5 in the supplemental material). They showed a significantdecrease in N nucleotides relative to those of WT embryos, definedboth as N⁺ DJ_H frequencies (13%; P < 0.001) and as the lengthsof N stretches per N⁺ sequence (1.6 ± 0.4; P < 0.01)(Fig. 5A). Interestingly, the group of Polµ^–/–DJ_Hs included many embryos obtained from matings of mutant maleswith RAG2^–/– females to eliminate the maternal contribution.Only 2 out of 26 of these DJ_Hs showed N nucleotides (P <0.05 with respect to the equivalent group of WT embryos derivedfrom RAG2^–/– females).

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FIG. 5. Differential features of Polµ^–/– embryo DJ_H joints. (A) Frequencies of N⁺ DJ_H joints (left) and numbers of N nucleotides per N⁺ sequence (right) in WT (thickly hatched) and Polµ^–/– (thinly hatched) embryos. (B) Frequencies of usage of SSH⁺ DJ_H joints (left) and of $≥$ 3-nt-long SSH (right) in WT and Polµ^–/– embryos. (C) Deleted nucleotides per DJ_H joint in WT and Polµ^–/– embryos and in newborns (a logarithmic scale is used in the x axis on the left). The arrows indicate the mean values for each group. The distribution profiles of DJ_H joints related to the extents of nucleotide deletions in WT and Polµ^–/– embryos and in WT newborns as defined by Gaussian kernel density estimations are shown in the right histograms (a linear scale is used in the x axis). The vertical lines delimit the highly preserved DJ_Hs ( $≤$ 2 deleted nucleotides). (D) Frequencies of highly preserved DJ_H joints detected in perinatal pre-B/B, adult B1, and adult pre-B/B cells from healthy mice in an extensive literature review (n = 1,572 DJ_H joints from 27 series in 19 papers). Each bar corresponds to an independent series of DJ_Hs. The numbers above the bars refer to the original articles, which are listed in Table S2 in the supplemental material. The horizontal lines denote the mean of each group.

Embryo Polµ^–/– DJ_Hs relied more on the utilizationof SSH for joining than those of WT embryos (P < 0.01), andthe former SSH tended to be distinctively longer (39% and 19%of Polµ^–/– and WT embryo SSH, respectively,were >3 nt long; P = 0.081) (Fig. 5B). Also, fewer joint-distalSSH were used among Polµ^–/– than WT embryoDJ_Hs, which therefore more closely resembled WT newborn DJ_Hs(P = 0.09; data not shown). The average numbers of nucleotidedeletions did not differ for WT and Polµ^–/–embryo DJ_H joints. However, we observed that a subset of highlypreserved DJ_H joints ( $≤$ 2 deleted nucleotides), which accountedfor 17% and 25% of WT embryo and newborn DJ_Hs, respectively,were selectively depleted from Polµ^–/– embryoDJ_Hs (P < 0.05 and P < 0.01 compared to WT embryos andnewborns, respectively) (Fig. 5C, left). The distribution profilesof DJ_H nucleotide deletions further revealed that the WT embryoDJ_Hs showed a bimodal pattern, with peaks centered on 5- and11-nt-long deletions, whereas newborn DJ_Hs were predominantlylimited to the less deleted subset. The Polµ^–/–DJ_H profile resembled that of newborn DJ_Hs in the rarity ofhighly deleted joints, although it differed from the other twoin the selective absence of highly preserved joints (Fig. 5C,right). To elucidate whether the latter sequence subset wasa consistent feature of every DJ_H repertoire, we undertook anextensive review of published DJ_H/VDJ_H joints from WT adultand perinatal mice (n = 1,572 DJ_H joints). All the analyzedseries included a fraction of highly preserved DJ_H joints (15.2%± 1.5%) (Fig. 5D) (the original references are shownin Table S2 in the supplemental material), suggesting that highlypreserved DJ_Hs represent an intrinsic result of the V(D)J rearrangementprocess and that the activity of Polµ is essential fortheir generation. Taken together, the above-mentioned findingsshow that Polµ participates in DJ_H coding-end ligationin the mouse embryo by (i) introducing a limited number of Nnucleotides, (ii) filling in small gaps in a template-dependentmanner, and (iii) contributing to recombination with end-distalSSH sites of the D sequence.

DISCUSSION

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DISCUSSION

The Ig V region repertoires differ between the perinatal andadult periods of life, with the former preferentially expressingD-proximal V_H genes (1, 69), showing distinct recombinationsites (41), and, more importantly, lacking the diversifyingpotential of the N-nucleotide-producing TdT polymerase (8, 27,32, 44, 55, 57). In the absence of TdT activity, the usage ofgerm line SSH in the recombination between most coding genepartners is predominant. B-lymphoid differentiation, however,starts much earlier, in the midgestation mouse embryo (14, 19,51, 73). We have shown here that the embryo DJ_H joints haveunusual characteristics, such as (i) short stretches of N nucleotidesin the absence of detectable TdT expression, (ii) reduced utilizationof SSH in embryo N^– DJ_Hs compared to those of newbornN^– DJ_Hs, and (iii) frequent DJ_Hs rearranging with joint-distalD-gene SSH, which results in extensive DJ_H nucleotide deletions.The embryo N⁺ DJ_Hs were also found among those recombined inORF2 (which are strongly counterselected for in the adult) and,more importantly, in DJ_H RAG2^+/– embryos derived fromRAG2^–/– mothers, demonstrating that they were ofembryonic origin. Most adult-derived N nucleotides are introducedby TdT (30, 42), although a few N nucleotides remained in TdT^–/–mice and low levels of N⁺ V(D)J joints were also detected innon-TdT-expressing perinatal life periods (21, 63). In agreementwith others, we could not detect any TdT transcript signal inembryo hematopoietic cells, which might account for the N nucleotidesfound. Also, when a limited pool of TdT^–/– embryoand adult DJ_Hs (n = 23 and 13, respectively) were sequenced,N nucleotides were not found in the adult, whereas a subsetof N⁺ DJ_Hs were detected in the embryos, supporting the TdTindependence of the embryo DJ_H N additions (M. A. R. Marcosand M. L. Gaspar, unpublished data). There is evidence of selectionagainst the utilization of N nucleotides in neonatal B and Tcells (7, 11, 12), as well as of positive selection for N^–IgHs among marginal-zone B cells (10) and for N^– public(26) and highly shared T-cell receptor β chains (38), suggestingthat the levels of N⁺ V(D)J joints under TdT^– conditions(as is the case in the perinatal periods) might even be higherin the absence of antigen selection and that there is room forthe activities of other polymerases (30).

The embryo N^– DJ_H joints did not show a predominant usageof SSH-related recombination sites (as happened in the newbornDJ_Hs), suggesting that SSH were initially used stochasticallyand that only afterwards were SSH⁺ DJ_H joints preferentiallyexpanded, because SSH increased the efficiency of DJ_H rearrangementsand/or because SSH⁺ DJ_Hs were positively selected for, as previouslyproposed (59). Whereas most SSH⁺ newborn DJ_Hs used joint-proximalD nucleotides, thus preserving the genomic sequence, a significantfraction of embryo DJ_Hs ligated to joint-distal SSH, resultingin large germ line nucleotide deletions. These highly deletedDJ_H joints were infrequent in later ontogenic periods, but theyhave been described in Bcl-XL transgenic mice, suggesting thatB-cell precursors bearing "inefficient" V(D)J products werenormally eliminated by apoptosis (25). Interestingly, the fewV(D)J rearrangements rescued in DNA-PK-deficient mice also displayedsignificant nucleotide deletions that were joined in embryobut not in adult V-J ${lambda}$ 1 joints, implying that the NHEJ machineriesacting in the two stages of life are different (43, 66). Bothembryo and perinatal DJ_H⁺ B-cell precursors might representindependent compartments in which distinctive NHEJ complexesact. Alternatively, we are tempted to suggest that neonatalDJ_Hs are a fraction selected from those emerging in the postgastrulationmouse embryo, after negative selection of most ORF2-expressingN⁺ and highly deleted DJ_H joints and/or promotion for ORF1⁺SSH⁺ DJ_Hs. As the embryo DJ_H patterns (particularly the presenceof N nucleotides) were already undetectable in E16 DJ_Hs whenIgH and the ${lambda}$ 5/V preB-encoded surrogate L chain were present(51, 63), it is possible that differential pairing of the emergingIgHs with the surrogate L chain is involved in filtering outthe embryos’ "inadequate" IgH chains during late fetalperiods (54, 72) and subsequently gives rise to a select perinatalIgH repertoire.

The processing of V(D)J coding joints can be sustained by thecore NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligaseIV) (34), but the family X polymerases (Polµ, Pol ${lambda}$ , andTdT) further contribute, especially when the nick leaves 3'overhangs, which are good substrates for them (45, 49, 58).There is a gradient of template dependence in the activitiesof X polymerases (56, 58), as Pol ${lambda}$ requires an extensive alignmentof the ends to exclusively catalyze template-directed gap filling(60, 61), whereas the lymphoid-specific TdT activity is incompatiblewith the template strand due to interference with its rigidloop 1 domain (20) and so is restricted to catalyzing N additionsin V(D)J reactions. In contrast, Polµ has a more flexibleloop 1, which allows ambivalent strategies (39): (i) it preferentiallyinserts templated nucleotides at small gaps created from overhangswith minimal or even null complementarities and (ii) it mayalso add nucleotides in a terminal-transferase-like fashionto create a "connector" that provides de novo microhomology.The latter N nucleotides would then be initially masked but,if elongated with a second N addition or with a templated insertionor used for promiscuous ligation, would be detected as bonafide N nucleotides. Polµ is biased to insert pyrimidinesversus purines, but it is important to consider that any untemplatedinsertion of Cs and Gs would be favorably selected for uponend joining, due to the greater strength of G·C thanA·T pairs. By analyzing Polµ^–/– embryos,Polµ was found to be responsible for most N nucleotidesdetected in the embryonic WT DJ_Hs, although a few N nucleotidesstill remained, which might be accounted for by either Pol ${lambda}$ (alsoexpressed in the embryo) or other activities. Some in vitroinsights into the Polµ potential to catalyze N nucleotides(including the experiments shown here) have been produced; theresults obtained in the embryo DJ_H joints represent (to ourknowledge) the first in vivo evidence of the TdT-like Polµactivity. Polµ was also required for the formation ofhighly preserved DJ_H sequences (<3 deleted nucleotides),which are always present in every DJ_H repertoire observed inhealthy mice. Another feature of Polµ^–/– embryoDJ_Hs was the relative reduction in sequences with large nucleotidedeletions, which were observed in WT embryos. These simultaneousdecreases in highly deleted and in highly preserved DJ_Hs (plusreduced N additions) in Polµ^–/– embryos explainedwhy the mean deletion rates were similar in WT and Polµ^–/–embryo DJ_Hs, despite the fact that the latter became a morehomogeneous group. Finally, more and larger recombination-relatedSSH were used in the Polµ^–/– than in the WTembryo DJ_Hs, likely representing a backup mechanism, as is observedwhen the classical NHEJ process is altered (15). It has beenreported that the VJ ${kappa}$ joints of adult Polµ^–/–mice showed increased nucleotide deletions (4). The authorsdid not observe any change in V(D)J_H processing, presumablydue to insufficient Polµ expression in IgH-rearrangingadult BM pro-B cells. The apparent discrepancy with our workmay be accounted for by the different ontogenic origins of thesamples and, in particular, by the higher expression of Polµin embryo DJ_H-rearranging precursors, which could thus reachthe threshold level required to participate in DJ_H joining.

A scenario for the variations in the V(D)J rearrangement processduring ontogeny might be proposed as follows. RAG activity isswitched on in the postgastrulation mouse embryo when it inducesthe first DSBs at immune-specific loci. During early embryoontogeny, somatic cells proliferate very actively and are notablysensitive to genotoxic stresses. Subsequently, robust machineriesare required to ligate imprecise and highly processed codingjoints in order to preserve cell viability and genome stability(35, 64). The evolutionarily ancient X polymerase Polµ,which is part of the cellular response to DSBs (9, 50), is wellequipped to connect these embryo DSBs and to avoid collateralgenomic damage (Fig. 6). First, Polµ can use templatednucleotide insertion to allow complete or almost complete conservationof the germ line sequence (Fig. 6, middle left) (17, 58). Ingood agreement with this, we observed that highly preservedDJ_H joints disappeared from Polµ^–/– embryos.Secondly, Polµ's terminal-transferase activity can selectivelytarget short DSB overhangs arising after hairpin nicking andopening (2-nt-long overhangs are the shortest nonblunt ends)(47), where it can introduce a few Ns, which may contributeto the pairing of DNA ends (Fig. 6, left) (17), particularlyin the cases of C and G additions. Alternatively, after endbridging by Polµ, N nucleotides could also be inserteddue to an imprecise orientation of the templating base thatleads to an insertion error/point mutation, ready for ligation(Fig. 6, middle right). Finally, exhaustive processing of DNAends, resulting in deletion and extensive 3' overhangs, canstill be handled by Polµ through its primer-realigningcapacity (67, 75) and avidity for internal 5' P, subsequentlypromoting pairing to internal SSH sites (16) at the cost ofgerm line sequence losses (Fig. 6, right). Challenged by strongevolutionary pressures to resolve cell-stressing DSBs in theearly embryo, Polµ thus may use all its skills to repairthe coding ends, even if many of the resulting products are"useless" and will be counterselected for later on. During thelast third of gestation, N^– RF1⁺ DJ_H/VDJ_H rearrangements,which preferentially used genome-preserving joint-proximal SSHs,selectively expanded, a process that may be secondary to changesin the NHEJ complexes used and/or to selection for pairing tosurrogate light-chain and/or (self) antigens. Both in the newbornand in the adult, Polµ will concentrate on the preservationof germ line coding ends by filling up small nucleotide gapsat those DSBs where it is recruited (IgL chain-encoding genesin the adult BM) (67, 75). The most recently evolved TdT willthen be upregulated in adult pro-B cells, and its exclusivetemplate-independent activity will be devoted to the generationof the extensive junctional diversity of IgH antibody chains.Finally, Polµ and TdT are not only compartmentalized duringontogeny and in different lymphoid differentiation stages, theyalso target distinct DSB DNA ends related to the extent of the3' overhangs produced by the nicking step, an event that mightalso influence the V(D)J repertoires during ontogeny.

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FIG. 6. A model for nucleotide insertions catalyzed by Polµ during embryonic DJ_H rearrangements. The Polµ structure (in gray) is depicted as two ellipses, the large one representing the polymerization core and the small one indicating the 8-kDa domain, which has a single-stranded DNA binding cleft for the 5' P binding site. The triangle represents mobile loop 1 of Polµ (39, 56). The deoxynucleotide triphosphate (dNTP) binding site is represented by a square (magenta). The hatched areas are "out-of-frame" J segments. (a) Germ line genomic region with D and J segments of the IgH chain. (b) RAG-induced hairpinned coding ends at both D and J segments. A nicking site is indicated by a small triangle at each end. (c) After nicking by Artemis-DNA-PK, a 3' overhang (2 nt in this case), which contains a P nucleotide, is generated at both ends. An internal 5' phosphate is indicated by a small circle. (d) Binding of Polµ to DNA ends can occur in two different ways: an enzyme-DNA complex can be formed in which the 3' protruding end is oriented as a primer strand whose primer terminus (P nucleotide) is close to the dNTP binding site (left); alternatively, the 5' P of one DNA end is bound by the Polµ 8-kDa domain next to the dNTP binding site (center and right). (e to j, left) Terminal-transferase N addition occurs before end joining (e). The inserted N nucleotide can serve as a connector for end joining if it is complementary to the other DNA end and the synapsis is stabilized by loop 1 (f). The N nucleotide, which is now inapparent as a template-independent insertion, is further extended by insertion of a templated dXTP (g). Once this strand is ligated, Polµ is adjusted to the remaining gap, coupled to a conformational change of loop 1 (h). After gap filling and ligation (i), the end joining is completed and produces an "out-of-frame" J segment (hatched area) due to the two extra P nucleotides added at the junction. An "in-frame" product (shown below) could be obtained by the addition of two N nucleotides before the end-joining step. In this case, one of the Ns would be evident (j). (f to j, middle left) Polµ's loop 1-mediated synapsis without requiring N nucleotides. If the two P nucleotides are complementary, their extension (X) will be template directed at both gaps, and the resulting product will not include N nucleotides (an "out-of-frame" J segment; one P nucleotide added). (f to j, middle right) If the two P nucleotides are not complementary and the templating base (located in front of the dNTP binding site) is not properly adjusted, Polµ can add N nucleotides. One or more N nucleotides could be immediately ligated and detected in the final recombination product. (c to j, right) When the ends are heavily processed and have large single-stranded overhangs, Polµ can bind the internal 5' P and attract a second 3' overhang to its vicinity, based on its capacity to accept microhomologies. After gap filling and further processing of the flapped strand, a largely deleted product is obtained that could contain either "out-of-frame" or "in-frame" J segments.

ACKNOWLEDGMENTS

This work was supported by grants SAF2007-65265, BFU2006-14390/BMC,and CSD2007-00015 from the Ministry of Education of Spain; bythe Fundación Mutua Madrileña; and by S-SAL-0304-2006from the Community of Madrid. The Centro de BiologíaMolecular Severo Ochoa receives institutional funding from FundaciónRamón Areces. B.G.-L. and N.S. are recipients of fellowshipsfrom Instituto de Salud Carlos III, and G.T. has a fellowshipfrom the Spanish Ministry of Science and Technology.

We thank Fernando Martínez and Mario Alía forexpert technical assistance, David Abia for statistical support,and Susana Morales and Daniel Lucas for contributions in theearly stages of the work.

FOOTNOTES

* Corresponding author. Mailing address: Centro de Biología Molecular, Campus de Cantoblanco, C. Nicolás Cabrera 1, 28049 Madrid, Spain. Phone: 34 911964672. Fax: 34 911964420. E-mail: marmarcos{at}cbm.uam.es

Published ahead of print on 22 December 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

M.A.R.M. and M.L.G. contributed equally as senior leaders ofthe research.

REFERENCES

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