Biallelic, ubiquitous transcription from the distal germline Ig␬ locus promoter during B cell development Rupesh H. Amina,1, Dragana Cadoa, Hector Nollaa, Dan Huanga, Susan A. Shintonb, Yan Zhoub, Richard R. Hardyb, and Mark S. Schlissela,2 aDepartment of Molecular and Cell Biology, 439 Life Science Addition, University of California, Berkeley, CA 94720; and bFox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 Edited by Frederick W. Alt, Harvard Medical School, Boston, MA, and approved November 6, 2008 (received for review September 8, 2008) Allelic exclusion of Ig gene expression is necessary to limit the receptor editing of the Ig␬ locus (6). If the preferred allele is number of functional receptors to one per B cell. The mechanism dictated, it is unclear how the second allele becomes a substrate underlying allelic exclusion is unknown. Because germline tran- for recombination if the first fails to encode a suitable protein. scription of Ig and TCR loci is tightly correlated with rearrange- Similarly, if recombination is rare and stochastic, it is unlikely ment, we created two novel knock-in mice that report transcrip- that a cell would ever recombine both alleles; yet a significant tional activity of the J␬ germline promoters in the Ig␬ locus. fraction of splenic B cells contain two rearranged Ig␬ alleles (6). Analysis of these mice revealed that germline transcription is All rearranging loci undergo sterile or germline transcription. biallelic and occurs in all pre-B cells. Moreover, we found that the Sterile transcripts originate from promoters upstream of the two germline promoters in this region are not equivalent but that recombining segments in a developmental and tissue-specific the distal promoter accounts for the vast majority of observed manner that correlates tightly with recombination of the asso- germline transcript in pre-B cells while the activity of the proximal ciated loci (7, 8). A recent report demonstrated that germline promoter increases later in development. Allelic exclusion of the transcription through a cluster of J␣ segments is necessary for Ig␬ locus thus occurs at the level of rearrangement, but not their recombination (9). Insertion of a transcription termination germline transcription. sequence into the J␣ cluster prevented local V␣-to-J␣ rearrange- ment. These workers concluded that RNA polymerase II transit allelic exclusion ͉ knock-in mouse across the recombining segments recruits chromatin modifica- tions that make the locus accessible to the recombinase. The J␬ he V(D)J recombinase and various DNA repair proteins cluster of gene segments contains two germline transcript pro- Tcatalyze the assembly of Ig heavy- and light-chain variable moters, located approximately 100 bp and 3.5 kb upstream of the region exons from dispersed gene-segments during B cell devel- J␬1 gene segment (10–12). Deletion of both promoters abolishes opment (1). The presence of two alleles for each gene and two ␬ locus recombination in AMuLV transformed pro-B cell lines, light chain loci (Ig␬ and Ig␭), suggests that, in theory, each B cell although the identical experiment has not been done in mice nor could produce eight different antibody specificities. However, as has either promoter been deleted individually (13, 14). Although postulated by Burnet 50 years ago (2), the vast majority of B cells germline transcription appears necessary for recombination, it is are monospecific. The underlying basis for the ‘‘one cell, one unclear if it is sufficient. If germline transcription is sufficient to receptor’’ phenomenon is the allelic and isotypic exclusion of Ig target the recombinase, monoallelic expression of germline gene expression such that the vast majority of mature B cells transcripts may underlie antigen receptor allelic exclusion. How- possess only one functional heavy and one functional light chain ever, a previous report demonstrated by single cell RT-PCR and gene rearrangement. Despite intensive effort, the mechanism RNA FISH that germline transcription of the J␬ cluster is underlying allelic exclusion is unknown. biallelic (15). This is in contrast to our report of variegated, Two general models have been proposed to account for allelic predominantly monoallelic expression of this locus (5). Because exclusion (3). The first is an instructive model in which an event these results are at odds with one another, it remains unclear how early in development differentially marks the two alleles such the J␬ germline transcripts are expressed and how this might that one allele is the preferred substrate for recombinase activity. regulate locus recombination. Once established, this differential mark propagates in a clonal To further probe the mechanism of allelic exclusion and its manner. The second model proposes a stochastic mechanism relation to germline transcription, we created two new knock-in where each allele is equally likely to recombine, but the prob- mouse strains that report transcriptional activity of the germline ability of recombination is low so that the percentage of cells Ig␬ locus. We found, contrary to expectation, that germline with two functional rearrangements is vanishingly small. In transcription of the J␬ cluster is biallelic and occurs in all pre-B support of the first model, it was observed that the two mouse cells. In reconciling this data with previous literature, we dis- Ig␬ alleles replicate asynchronously in S-phase beginning at a covered that the two J␬ germline promoters in the Ig␬ locus are very early stage of development (4). However, it has not been proven conclusively that the early replicating allele is the pref- erential target of recombination, nor what other marks might be Author contributions: R.H.A., D.C., R.R.H., and M.S.S. designed research; R.H.A., H.N., D.H., clonally propagated to dictate allele-specific recombination. In S.A.S., Y.Z., R.R.H., and M.S.S. performed research; R.H.A., R.R.H., and M.S.S. analyzed data; favor of the second model, we previously reported that a GFP and R.H.A. and M.S.S. wrote the paper. cDNA knocked-in to the mouse Ig␬ locus is expressed in only The authors declare no conflict of interest. 1–5% of pre-B cells (5). As germline transcription is tightly This article is a PNAS Direct Submission. correlated with recombination, we interpreted this to indicate 1Present address: Fred Hutchison Cancer Research Center, Seattle, WA. that only a small fraction of unrearranged Ig␬ alleles are 2To whom correspondence should be addressed. E-mail: [email protected]. transcriptionally active and therefore suitable targets for the This article contains supporting information online at www.pnas.org/cgi/content/full/ recombinase at any given time. Neither model fully accounts for 0808895106/DCSupplemental. observed recombination frequencies or patterns, in particular © 2008 by The National Academy of Sciences of the USA 522–527 ͉ PNAS ͉ January 13, 2009 ͉ vol. 106 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808895106 Downloaded by guest on October 1, 2021 A Distal germline Proximal germline transcript transcript Vκ cluster Jκ iEκ Cκ Distal germline transcript hCD4 ires YFP Cκ B CLP Fr. A Fr. B&C Fr. C' Fr. D Fr. E Fr. F 10 200 15 5 150 8 15 150 4 150 0.31% 3.03%10 5.12% 90.4% 91.5% 84.5% 51.6% 6 3 100 10 100 100 κ 4 d GT-hCD4 2 5 50 5 50 50 2 1 0 0 0 0 0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 hCD4 6 12 200 15 12 200 150 9 9 150 150 4 91.2% 85.6% 0.53% 2.53%10 1.09% 84.7% 96.4% 100 κ 6 6 100 100 C -iYFP 2 5 50 3 3 50 50 Number of cells 0 0 0 0 0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 YFP C T1 T2 T3 Fo MZ B-1 15 20 600 1200 60 50 15 40 10 900 58.8% 59.8% 54.5% 49.5%400 50.1%40 39.4% 30 10 κ 600 d GT-hCD4 20 5 200 20 5 300 10 0 0 0 0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 hCD4 80 60 1200 30 4000 150 60 900 95.9% 96.6%40 90.9%3000 82.6% 79.6% 81.8% 20 100 40 600 κ 2000 C -iYFP 20 10 50 20 1000 300 Number of cells 0 0 0 0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 YFP Fig. 1. Marker protein expression in d␬GT-hCD4 and C␬-iYFP knock-in mice. (A) Schematic of the d␬GT-hCD4 and C␬-iYFP knock-in mutations at the Ig␬ locus. Both the proximal and distal germline transcript promoters are shown as arrows. Previously defined splicing patterns of both transcripts are depicted as dashed lines. The hCD4 cDNA is followed by an SV40 intron and polyA sequence. Black triangles represent the positions of loxP sites remaining in the locus after Cre recombinase- mediated deletion of a neomycin resistance gene. (B) Flow cytometric analysis of hCD4 or YFP marker expression in developing bone marrow B cells from heterozygous d␬GT-hCD4 or C␬-iYFP knock-in mice. Harvested bone marrow was labeled with antibodies to delineate B cell developmental subsets and mark hCD4-expressing cells (in d␬GT-hCD4 mice only).