Antisense Intergenic Transcription Precedes Igh D-to-J Recombination and Is Controlled by the Intronic Enhancer E{micro}

V(D)J recombination is believed to be regulated by alterationsin chromatin accessibility to the recombinase machinery, butthe mechanisms responsible remain unclear. We previously proposedthat antisense intergenic transcription, activated throughoutthe mouse Igh V_H region in pro-B cells, remodels chromatin forV_H-to-DJ_H recombination. Using RNA fluorescence in situ hybridization,we now show that antisense intergenic transcription occurs throughoutthe Igh D_HJ_H region before D-to-J recombination, indicatingthat this is a widespread process in V(D)J recombination. Transcriptioninitiates near the Igh intronic enhancer E_µ and is abrogatedin mice lacking this enhancer, indicating that E_µ regulatesD_H antisense transcription. E_µ was recently demonstratedto regulate D_H-to-J_H recombination of the Igh locus. Together,these data suggest that E_µ controls D_H-to-J_H recombinationby activating this form of germ line Igh transcription, thusproviding a long-range, processive mechanism by which E_µcan regulate chromatin accessibility throughout the D_H region.In contrast, E_µ deletion has no effect on V_H antisenseintergenic transcription, which is rarely associated with D_Hantisense transcription, suggesting differential regulationand separate roles for these processes at sequential stagesof V(D)J recombination. These results support a directive rolefor antisense intergenic transcription in enabling access tothe recombination machinery.

INTRODUCTION

Top

INTRODUCTION

In order to generate the primary repertoire of immunoglobulin(Ig) and T-cell receptor (TCR) molecules, antigen receptor lociundergo variable, diversity, and joining [V(D)J] recombinationin B and T lymphocytes. Recombination is catalyzed by a recombinasecomplex containing the protein products of the recombinase-activatinggenes Rag1 and Rag2 (28). Within precursor lymphocytes, thisprocess is strictly lineage specific, with heavy (Igh) and light(Ig ${kappa}$ and Ig ${lambda}$ ) immunoglobulin loci fully recombining only in Blymphocytes and T-cell receptor loci (Tcra, Tcrb, Tcrg, andTcrd) recombining only in T cells. Further, within lineages,loci are recombined in a precise order. Recombination of theIgh locus is the earliest step in the generation of the matureantibody repertoire in B lymphocytes. The Igh locus of the C57BL/6mouse spans 3 Mb and comprises 195 V_H genes spanning 2.5 Mb,10 D_H genes ( $~$ 60 kb), 4 J_H genes (2 kb), and 8 constant (C_H)genes (200 kb) (31, 68). D_H-to-J_H recombination occurs on bothIgh alleles before V_H-to-DJ_H recombination takes place (16).

Lineage and stage specificity of V(D)J recombination are regulatedby differential chromatin accessibility to the RAG proteins.Several mechanisms may contribute, but their relative importanceis still unclear. The first process discovered was germ linetranscription, which occurs in all antigen receptor loci acrossgene segments competent for recombination (34). This transcriptionwas termed "sterile" or "germ line" to distinguish it from codingV(D)J transcription. In the Igh locus, the earliest germ linetranscripts detected occur before D_H-to-J_H recombination andinitiate from two regions: the intronic enhancer E_µ (I_µtranscript) (35) and a promoter, PDQ52, immediately upstreamof the most 3' D_H gene segment, DQ52 (_µ0 transcript) (60).Following D_H-to-J_H recombination, the DJ_H gene segment producesD_µ transcripts (51) and sense germ line transcriptioninitiates over the V_H genes (17, 67). The discovery of V_H genegerm line transcription formed the basis of the accessibilityhypothesis, which proposed that lineage and stage specificityof recombination are regulated by differential chromatin accessibilityof antigen receptor gene segments to the recombinase machinery,with germ line transcription associated with open chromatin(58, 67). However, a function for V_H germ line transcriptionhas not been demonstrated, and it has been argued that it maybe a secondary effect of the V_H gene promoters becoming accessiblefor V_H-to-DJ_H recombination.

We have recently provided evidence in support of a functionalrole for germ line transcription with the discovery that anadditional process, intergenic antisense transcription, notcontrolled by V_H gene promoters, occurs throughout the V_H regionbefore V_H-to-DJ_H recombination. We proposed that this large-scaletranscription remodels the V_H region to facilitate accessibilityfor V_H-to-DJ_H recombination (8).

Intergenic transcription has been proposed to drive throughrepressive chromatin, recruiting remodeling factors, thus facilitatingfurther chromatin opening over genes (12). In several loci,intergenic transcription delineates domains of modified chromatinthat surround active genes and their regulatory elements (7,21, 25). RNA polymerase II is associated with, or recruits,a wide range of chromatin-remodeling and histone-modifying factors(14, 65, 66). Furthermore, transcription triggers histone turnoverand the deposition of variant histone H3.3, enriched with activemodifications (54). Collectively, these activities suggest severalmechanisms by which the processing activity of the elongatingRNA polymerase II complex can achieve chromatin accessibility(42, 47).

The process of intergenic transcription may therefore directthe changes in chromatin structure that precede V(D)J recombination.In the IgH V region, these include acetylation of histones H3and H4, markers of accessible chromatin (15, 29, 38); histoneH3.3 exchange (30); and methylation of H3 lysine 27 (H3-K27)(59). Indeed, it was recently demonstrated that disruption ofintergenic transcription in the J ${alpha}$ domain of the mouse TCR ${alpha}$ locusresults in failure both to methylate H3 lysine 4 and to recombineseveral corresponding J ${alpha}$ genes (1), demonstrating a requirementfor intergenic transcription to effect V(D)J recombination.In addition to localized changes, several large-scale changesin nuclear configuration occur, including repositioning of theIgh loci away from the nuclear periphery (32) and Pax-5-dependentcontraction of the V_H region, via higher-order looping of chromatin(23, 32, 53).

In the D region, increased DNase I sensitivity (15, 37), enrichmentof acetylated histones H3 and H4, H3K4 methylation, and nucleosomeremodeling over D_H and J_H genes occurs in pro-B cells poisedfor D_H-to-J_H recombination (15, 37, 43). Overall, while it isevident that several processes play critical roles in the regulationof V(D)J recombination, the precise role of each remains unclear.

It also remains to be clarified which regulatory elements directV(D)J recombination of the Igh locus. It was originally proposedthat the intronic enhancer E_µ regulated V_H-to-DJ_H recombination(52, 55). However, more recent studies by ourselves and othershave shown that targeted deletion of E_µ causes a defectin D_H-to-J_H recombination, indicating that E_µ regulatesthis process primarily (2, 49). Thus, previously observed defectsin V_H-to-DJ_H recombination are likely to be secondary to thisearlier defect, and a V region-specific recombination controlelement has not yet been found. Although the function of E_µin V(D)J recombination is now known, its mechanism of actionremains unclear, and much remains to be understood about thechromatin regulation of the D_H region before D_H-to-J_H recombinationin vivo.

Our discovery of intergenic antisense transcription over theIgh V region before V-to-DJ recombination raised the questionof whether similar transcriptional processes precede other V(D)Jrecombination events. Here we ask whether germ line transcriptionoccurs over the D_H region and whether this precedes and is functionallyassociated with D_H-to-J_H recombination.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

Mice. Rag1^–/– mice (57), backcrossed to a C57BL/10 background,were kindly provided by M. Turner. C57BL/6 wild-type (WT) miceand timed matings were from the Small Animal Barrier Unit, BabrahamInstitute. PDQ52 and E_µ gene-targeted mice were as describedpreviously (2). Animal work was performed under project licensePPL 80/1644, in compliance with Home Office guidelines.

Sorting of B-lymphocyte populations. CD19⁺ B lymphocytes from Rag1^–/– bone marrow (BM)were sorted for RNA by positive selection using anti-CD19 magneticaffinity cell sorting (MACS) magnetic beads (Miltenyi Biotech;purity, >80% CD19⁺). B220^– interleukin 7R (IL-7R)⁺cells (common lymphoid progenitors [CLPs]) were fluorescence-activatedcell sorter (FACS) sorted directly from WT BM without priorenrichment. We sorted B220⁺CD19⁺ Rag1^–/– BM B cellsfor RNA fluorescence in situ hybridization (RNA FISH) directlyby FACS. The following antibodies, obtained from BD PharMingen,were used: phycoerythrin-labeled, allophycocyanin (APC)-labeled,or biotinylated anti-B220 (RA3-6B2); fluorescein isothiocyanate(FITC)-, APC-, or peridinin chlorophyll-cy5.5-labeled or biotinylatedanti-CD19 (1D3); and biotinylated anti-IL-7R (B12-1). The purityof each cell fraction was always >90%. Cells were washedtwice in phosphate-buffered saline and then applied to poly-L-lysine-coatedslides (Sigma) and fixed with 3.7% formaldehyde (Merck).

Fetal liver B cells were isolated by negative MACS selectionusing a biotinylated anti-Ter119 antibody (BD Pharmingen). B220⁺CD19⁺cells were then sorted from the negative fraction by FACS usingthe same antibodies described above. These cells were then usedto make either RNA or slides for RNA FISH as described above.B220⁺ cells were sorted from BM of Rag-deficient mice harboringeither WT, PDQ52^–/–E^+/+, or P-E^– alleles,using biotinylated anti-B220, followed by streptavidin-conjugatedMACS beads.

Reverse transcription-PCR analysis. Total RNA was extracted from BM cell populations and cell linesusing an RNeasy kit (QIAGEN) according to the manufacturer'sprotocol, except for total RNA from Rag-deficient mice harboringWT, PDQ52^–/–E^+/+, or P-E^– alleles, which wasisolated using TRIzol (Invitrogen Life Technologies). The RNAwas treated with RNase-free DNase I (1 unit per µg oftotal RNA; Promega) for 1 h at 37°C followed by repurificationusing a QIAGEN RNeasy kit according to the manufacturer's RNAcleanup protocol. Between 0.1 and 0.5 µg of total RNAwas reverse transcribed for 2 h using Superscript III (Invitrogen)at either 50°C, with 100 ng of random hexamers (AmershamPharmacia), or 55°C, with 2 pmol of specific reverse transcriptionprimer (see Table S1 in the supplemental material). Reversetranscription reactions were assembled at 55°C to reduceendogenous priming. Following reverse transcription, PCR wasperformed with the primers listed in Table S1 in the supplementalmaterial, using the following conditions: 30 to 40 cycles of30 s at 94°C, 30 s at the annealing temperature, and 30s at 72°C. Annealing temperatures varied according to themelting temperatures of the primer pairs (see Table S1 in thesupplemental material). Inverted gel images were sufficientto visualize products, with the exception of 81X (see Fig. 3),which was detected by Southern blotting using a ³²P-labeledgenomic DNA PCR product probe. DNA bands were excised, gel purifiedusing a QIAquick gel extraction kit (QIAGEN), and cloned intothe pGEM-T Easy vector (Promega) for sequencing. Real-time PCRwas performed in triplicate on cDNA reversed transcribed withrandom hexamers as previously described, except 1 µg oftotal RNA was used. PCRs were detected with SYBR green fluorogenicdye (Applied Biosystems) in an ABI PRISM 7000 sequence detectionsystem (Applied Biosystems). Cycling conditions were as follows:95°C for 10 min followed by 40 cycles of 95°C for 15s and 60 to 64°C (depending upon primer pair) for 1 min.The PCR primers used are listed in Table S1 in the supplementalmaterial. Relative quantification was performed after constructionof standard curves using serial dilutions of genomic DNA, andvalues were then normalized to J558.

View larger version (82K):
[in this window]
[in a new window]

FIG. 3. Semiquantitative reverse transcription-PCR analysis of D_H antisense and V_H genic germ line transcription through development. Top panel: Day (D)14.5, D15.5, and D16.5 fetal liver B lymphocytes. Bottom panel: Rag1^–/– CD19⁺ BM B lymphocytes, CLPs, and ex vivo thymocytes. Right margin, regions amplified; left margin, molecular sizes. –RT, without reverse transcriptase. ß-actin was used as a loading control. DSP and DFL16.1 antisense transcripts were detected as in Fig. 2. 81X and J558 were amplified by random-primed reverse transcription-PCR. The PCR primers were designed to amplify one (81X) or nine (J558) V genes. 81X products were subjected to blotting Southern and probed with a genomic DNA PCR product to aid visualization.

RNA FISH. Sorted cells fixed on slides were analyzed by two-color RNAFISH essentially as described previously (24), with the modificationslisted by Gribnau et al. (25). For three-color FISH, biotinylatedprobes were generated and detected with streptavidin Alexa 350(Molecular Probes), followed by a biotinylated goat antistreptavidinantibody (Cambio), followed by an additional layer of streptavidinAlexa 350. When Alexa 350 was used, DAPI (4',6'-diamidino-2-phenylindole)was omitted from the mounting media. Probes for FISH were generatedby PCR (primers are listed in Table S1 in the supplemental material),cloned into pGEM-T Easy vector (Promega), and verified by sequencing.Single-stranded cDNA probes for detection of either sense orantisense transcripts were generated by in vitro transcriptionusing a MAXIscript kit (Ambion) followed by reverse transcriptionwith Superscript II (Invitrogen) including either digoxigenic-11-dUTP(Roche), 2,4-dinitrophenol-11-dUTP (Perkin Elmer), or biotin-11-dUTP(Roche) in the reaction (11). In hybridizations, antisense probesdetected complementary sense transcripts, while sense probesdetected antisense transcripts. Signals were visualized andcaptured using Olympus BX40 and BX41 microscopes. Experimentswere performed two to three times, and at least 100 nuclei (twocolors) or 100 alleles (three colors) were counted each time.Total numbers of cells counted are shown in Table S2 in thesupplemental material.

RESULTS

Top

RESULTS

Germ line antisense and intergenic transcription occurs throughout the D_H region. We assembled in silico the entire D_H region of the C57BL/6 mouseIgh locus from available genomic sequences. Subsequently, theassembly of this region was reported elsewhere (68). Withinthis sequence, all D_H gene segments are oriented in the same(sense) direction relative to the constant region genes. TheC57BL/6 D_H region is organized as a tandem array of seven 4.7-kbrepeats, each repeat containing a DFL16.1 or DSP gene (Fig.1). The DSP repeats are almost identical to each other (95 to99%), while the DFL16.1 repeat is $~$ 85% identical to each of theDSP repeats. The most 3' D gene, DQ52, is separated from theDFL/DSP tandem array by $~$ 20 kb of unique sequence and has verydifferent flanking sequences, including the PDQ52 promoter/enhancer(33).

View larger version (18K):
[in this window]
[in a new window]

FIG. 1. DFL and DSP genes are located in a tandem array of $~$ 4.7-kb repeats. The sequence of the D_H region of C57BL/6 was aligned against itself using the DotPlot program from the Omiga package (Accelrys). The interspersed repeats within the sequence on the vertical axis were masked with X's using RepeatMasker (www.repeatmasker.org) to exclude these from the alignment, resulting in a discontinuous diagonal line, in particular in the DSP2.5-DQ52 intergenic region, which contains several interspersed repeats (gray boxes on x axis). Black arrows depict the $~$ 4.7-kb direct repeats containing the DSP and DFL16.1 genes. A sliding window of 30 bp was used; lines indicate $≥$ 90% identity. D_H genes are labeled in accordance with Ye (68). For clarity, the DST4 and P2-P6 genes are not depicted.

We first investigated germ line transcription of the DFL16.1/DSPregion, since this constitutes most of the D_H region and thevast majority of D_H gene usage in V(D)J recombination in C57BL/6mice (20, 68). We designed reverse transcription and nestedPCR primers to detect all the DSP genes equally in a singlereaction and a set to detect the DFL16.1 gene separately. Reversetranscription and PCR primers were located downstream of theD_H gene 3' recombination signal sequence (RSS) (Fig. 2A). Asthe 3' RSS is lost due to D_H-to-J_H recombination, only transcriptsoriginating from unrearranged D_H segments will be detected inWT mice. We first analyzed ex vivo CD19⁺ BM cells from Rag1^–/–mice (57) using a sensitive strand-specific reverse transcription-PCRassay (8). As these mice cannot perform V(D)J recombination,any D_H transcripts detected would be exclusively germ line inorigin.

View larger version (19K):
[in this window]
[in a new window]

FIG. 2. Detection of germ line transcription across the Igh D region. (A) Design of the D_H region germ line transcription reverse transcription-PCR assay. Reverse transcription antisense (to detect sense transcription) and sense (to detect antisense transcription) primers were designed 5' and 3' of the D_H gene RSS sequences. Nested primers were used for PCR. Similar schemes were used for intergenic regions. (B) Schematic of the DFL16.1-J1 sequence showing regions analyzed by reverse transcription-PCR. Not to scale. (C) Reverse transcription-PCR of Rag1^–/– CD19⁺ total RNA. Above lanes, with (+) or without (–) reverse transcriptase (RT). Right margin, regions amplified (shown in panel B); left margin, molecular sizes. DNA, genomic DNA positive control. (D) Sequencing of DSP genic and intergenic reverse transcription-PCR products. The number of reverse transcription-PCR products cloned and sequenced is shown for each D_H gene and intergenic region. (E) Real-time quantitative reverse transcription-PCR of Rag1^–/– CD19⁺ random-primed cDNA showing relative transcription levels for Hprt, DFL16.1, V_HJ558 intergenic, and V_HJ606 intergenic regions. The level of V_HJ558 intergenic transcription was arbitrarily set to 1.

We reverse transcribed total RNA from Rag1^–/– CD19⁺BM cells using random hexamers or gene-specific primers andperformed PCR on the cDNA using nested primers. In both randomand antisense-specific reverse transcription-PCRs, cDNA productsof the same size as those from genomic DNA were amplified forboth DSP and DFL, while no sense-specific products were amplified(Fig. 2C, i and ii). We did not detect any sense D_H germ linetranscription using two further sets of reverse transcriptionand nested PCR primers, confirming previous findings (3). However,as previously described (8), we detected sense, but not antisense,transcription over the I_µ intron and C_µ exons 2to 3 (Fig. 2B and C). We conclude that antisense, but not sense,transcription occurs over the germ line D genes of the Igh locus.

We next determined whether D_H antisense transcription was exclusivelyassociated with the genes or extended into intergenic regions.As the intergenic sequences between the DSP genes are 95 to99% identical, we were able to design one set of nested primersthat would amplify interspersed repeat-free regions betweeneach of the six DSP genes with equal efficiency (Fig. 2B, iii).Strand-specific reverse transcription-PCR showed an unsplicedantisense band of the expected size, indicating that antisensetranscription also occurred in intergenic regions (Fig. 2C,iii). We cloned and sequenced both the genic and intergenicreverse transcription-PCR products to find out whether the wholeDSP array was transcribed or if the observed transcription wasoccurring across only certain regions. All but one DSP geneand one intergenic region were represented, indicating thattranscription occurs across the entire DSP region (Fig. 2D).

We next investigated if the antisense transcription extendedoutside the DFL/DSP array using reverse transcription and nestedPCR primers located between the most 3' DSP gene, DSP2.5, andthe most J-proximal D gene, DQ52 (Fig. 2B, iv). A strong antisense-specificband, but no sense band, was detected, indicating that the DSP-DQ52intergenic region was also transcribed in the antisense orientation(Fig. 2C, iv). Taken together, the data indicate that antisenseintergenic transcription extends throughout the D_H region.

To gain insight into how abundant these transcripts are andhow their amounts compared to antisense transcripts we havepreviously detected in the V region, we performed quantitativereal-time reverse transcription-PCR. We compared transcriptionfrom the unique DF16 gene with two unique intergenic regions,the first within the J558 gene family which comprises the 5'half of the V region and the second from the J606 family inthe middle of the V region. The reverse transcription reactionwas performed with random hexamers so that the subsequent PCRscould be performed on the same sample. In all three cases, transcriptionhas previously been detected only from the antisense strand.DFL16 transcript amounts were intermediate between J558 andJ606, and variation was no greater than 2.5-fold among all threeregions (Fig. 2E), indicating that steady-state amounts of antisensetranscription over the D region are comparable to the V regionin Rag1^–/– CD19⁺ BM cells. Furthermore, amountswere within fourfold of the housekeeping gene, hypoxanthine-guaninephosphoribosyltransferase (HPRT), indicating that these transcriptsare relatively abundant.

Developmental regulation of D_H antisense transcription. We next sought to investigate the stage specificity of D_H antisensetranscription during B-lymphocyte development and V(D)J recombination.B lymphopoiesis initiates in the fetal liver with B-cell progenitorsappearing by day 12.5 (18). D_H-to-J_H recombination initiateson day 12 and increases rapidly on days 13 and 14 (19). Thus,fetal liver on day 14 and day 15 includes B cells that haveinitiated and those that have completed D_H-to-J_H recombination(26). Consequently, initiation of V_H-to-DJ_H recombination issimilarly asynchronous, and this is reflected by the reportedexpression of V_H germ line sense gene transcripts from day 14until day 16 (36). Thus, in fetal liver, D_H-to-J_H recombinationand V_H-to-DJ_H recombination occur in partially overlapping temporalwindows.

We first investigated steady-state transcript levels in BM progenitorB lymphocytes from Rag1^–/– (CD19⁺) and day 14.5,15.5, and 16.5 WT fetal liver B cells. Further, to obtain amore detailed picture of when D_H antisense transcription initiates,we also examined BM CLPs (IL-7R⁺ B220^–), which undergoa low level of D_H-to-J_H recombination (4). We analyzed thymusto study the association of D_H antisense transcription specificallywith D_H-to-J_H recombination, since up to 50% of thymocytes undergoD_H-to-J_H but are blocked from completing V_H-to-DJ_H recombination(9). We used brain as a negative control. By semiquantitativereverse transcription-PCR, we first analyzed expression of Vregion germ line transcripts. Random-primed reverse transcriptionfollowed by PCR to detect the largest J558 gene family in theD-distal end of the V region yielded two transcript bands, aspreviously shown for BM early B cells. The lower band correspondsto the spliced sense transcript, while the upper band includesboth antisense and unspliced sense primary transcripts (8).Both bands were present in fetal liver B cells, with littlevariation between days 14.5 and 16.5 (Fig. 3), confirming previousNorthern analysis (36). We found a similar developmental distributionof germ line transcripts from the D-proximal 7183 V gene family,albeit transcript levels were lower, due in part to PCR detectionof fewer genes (Fig. 3). Neither transcript was present in thethymus, consistent with the V region's being inaccessible inthymocytes (Fig. 3). Steady-state D_H antisense transcriptionlevels were similar to those in Rag1^–/– CD19⁺ cellsin fetal liver B-cell populations, with little change betweendays 14.5, 15.5, and 16.5 (Fig. 3). D_H antisense transcriptsare absent from brain (not shown) and low in CLPs but are readilydetectable in thymocytes (Fig. 3). Taken together, these resultssuggest that D_H antisense transcription initiates on Igh allelespoised for D_H-to-J_H recombination.

Reverse transcription-PCR was used to assess steady-state transcriptlevels in bulk populations. However, we sought to analyze D_Hantisense primary transcription on individual Igh alleles insingle cells to determine its association with the differentsteps of V(D)J recombination of the locus and thus provide cluesto its function. To do this, we used RNA FISH. We designed 3.5-kbDFL16.1 and 2.8-kb DSP2.x1 RNA FISH probes containing each geneand respective flanking region (Fig. 4A). As the DSP repeatsare >95% identical, it is likely that the DSP2.x1 probe detectstranscription of all DSP genes. As the DFL and DSP segmentsare highly homologous, it was also probable that the probeswould cross-hybridize. Therefore, for optimal detection efficiency,we combined the probes in all cases.

View larger version (31K):
[in this window]
[in a new window]

FIG. 4. Developmental regulation of D_H antisense transcription in fetal liver B lymphocytes. (A) Locations of RNA FISH probes: red, detected with Texas Red; green, detected with FITC. RNA FISH detection of D_H antisense intergenic and I_µ sense transcription in (B) CD19⁺ Rag1^–/– BM and (C) WT E15.5 fetal liver B lymphocytes. (D) RNA FISH detection of V_HJ558 antisense transcription in day 15.5 fetal liver B lymphocytes. (E) RNA FISH detection of V_HJ558 VDJ-rearranged (VDJ_H) transcription in day (D)16.5 fetal liver B lymphocytes. Similar signals were obtained for V_H7183 and V_HQ52. In all cases, nuclei were counterstained with DAPI (blue). (F) Quantitation of D_H antisense, V_HJ558 antisense, J558 VDJ_H, 7183 (81X) VDJ_H, and Q52 VDJ_H RNA FISH signals in CD19⁺ B lymphocytes from D14.5, D15.5, D16.5, and D17.5 fetal liver.

To first determine if we could detect D antisense intergenictranscription by RNA FISH, we analyzed CD19⁺ pro-B cells fromRag1^–/– mice. Sense or antisense D_H probes werecohybridized with a probe for I_µ, described previously(8). We used I_µ as a positive control, as it is transcribedfrom a high proportion of alleles (8) (Fig. 4A). We detectedD_H antisense signals in 37% of Rag1^–/– CD19⁺ pro-Bcells with I_µ signals. We did not detect any D_H sensesignals, in agreement with the reverse transcription-PCR results.D_H antisense signals were closely associated with or overlappingthe I_µ signals, as expected, given that the DSP/DFL regionis located just $~$ 24 kb from E_µ (Fig. 4B). We detected biallelicD_H antisense transcription in $~$ 10% of cells, indicative of thestochastic output of a biallelically expressed transcriptionunit transcribing at a low level (61). In conclusion, we wereable to detect D_H antisense transcription by RNA FISH in a highproportion of Rag1^–/– pro-B cells.

We next investigated D_H antisense intergenic transcription inday 14.5, 15.5, 16.5, and 17.5 fetal liver B220⁺CD19⁺ B-lymphocytepopulations (Fig. 4C). I_µ signals were detected in approximately60% of cells in each of these samples, indicating that a largeproportion of Igh alleles are active in fetal liver B lymphocytes.In day 14.5 and 15.5 populations, we detected D_H antisense signalsin comparably high proportions of cells (30% and 32% of cellswith I_µ signals, respectively). By day 16.5, the numberof B cells with D_H antisense signals was reduced to $~$ 18%, andthis number was further reduced to 7% by day 17.5 (Fig. 4F).Thus, D_H antisense transcription occurs over an extended timewindow during fetal liver B-lymphocyte development but is progressivelylost, probably as a result of ongoing V(D)J recombination (seebelow).

To further clarify how D_H antisense transcription relates toV(D)J recombination in fetal liver, we analyzed the appearanceof sense transcripts from VDJ_H-rearranged alleles. V_H sensegerm line transcripts are undetectable by our RNA FISH technique(8); thus, all V_H sense transcription signals detected originatefrom VDJ_H-rearranged alleles. We used probes to detect VDJ_H-rearrangedtranscription for the 3' V_H7183 (81X) and V_HQ52 families andthe large 5' V_HJ558 family (8). In day 15.5 fetal liver B lymphocytes,we detected VDJ_H signals for both V_H7183 and V_HJ558 in similarlylow numbers of cells ( $~$ 3% of cells with I_µ) (Fig. 4E and F).By day 16.5, signals for both families had increased to 10%,while at day 17.5, V_H7183 signals were found in 17% and V_HJ558signals in 21% of cells with I_µ signals. This distributionis as expected, given the greater number of J558 genes (31),tempered by a mild bias toward V_H7183 recombination in C57BL/6fetal liver (5). These results correlated well with a previousreport of Northern blot detection of J558 VDJ_H transcripts fromday 16.5 of development (36). Signals from the V_HQ52 familywere found in 6.5% and 8% of day 16.5 and 17.5 B lymphocytes,respectively (Fig. 4F). Thus, V_H-to-DJ_H recombination also occursasynchronously over an extended temporal window. Together, thesefindings show that by the time VDJ_H signals are seen in appreciablenumbers (day 16.5), the number of D_H antisense signals is greatlyreduced, consistent with D_H antisense transcription's beinglost as a result of ongoing V(D)J recombination.

Due to the overlapping time windows of D_H-to-J_H and V_H-to-DJ_Hrecombination, we had not yet determined the extent to whichD_H antisense intergenic transcription was associated with eachof these processes. To place it in context relative to the accessibilityof the V_H region, we next analyzed V_H antisense transcriptionin fetal liver. Using a probe to the V_HJ558 antisense transcriptsfrom the 5' half of the V region (8), we discovered that V_Hregion antisense transcription is present in a relatively highnumber of cells by day 14.5 (19% of cells with I_µ signals)(Fig. 4D and F). This proportion remains similar on day 15.5(18.5%) but is reduced by day 16.5 (12%) and further reducedby day 17.5 (6%). Thus, V_H and D_H antisense transcription processesalso overlap significantly within the same time window, albeittranscripts may occur on different alleles or indeed in differentcells.

The sharp reduction in both D and V antisense primary transcriptsbetween day 15.5 and day 16.5 and thereafter (Fig. 4F) contrastedwith steady-state transcript levels, which showed little variationbetween days 14.5 and 16.5 (Fig. 3). RNA FISH detects a windowof primary transcript generation and thus is exquisitely dependenton the presence of an actively transcribing locus and appropriatetarget sequence, which is lost due to ongoing V(D)J recombination.In contrast, steady-state levels reflect the balance betweengeneration and degradation of transcripts, and the relativelystable levels observed suggest low transcript turnover.

D_H antisense transcription initiates on germ line alleles and before V_H antisense transcription. To distinguish between these possibilities, we first determinedif D_H and V_H antisense transcription occurred on germ line allelesin day 15.5 fetal liver B lymphocytes using three-color RNAFISH. We cohybridized probes to detect I_µ, _µ0, andeither D_H (DSP and DFL) or V_H (J558) antisense transcription(Fig. 5A). The _µ0 probe is located between DQ52 and J1;since this region is removed by D_H-to-J_H recombination, thisprobe is a marker of unrearranged alleles. We detected I_µin blue, _µ0 in red, and D_H or V_H antisense transcriptionin green. We found that D_H antisense signals colocalized with_µ0 and I_µ in 36% of total alleles counted in day15.5 fetal liver B lymphocytes, while D_H antisense signals occurredwithout _µ0 on 17% of total alleles (Fig. 5A and C). Thus,70% of D_H antisense transcripts were associated with _µ0,indicating that D_H antisense transcription initiates on germline alleles in fetal liver. A total of 30% of alleles expressedD_H antisense transcripts without _µ0, suggesting that D_Hantisense transcription continues on DJ_H-rearranged alleles.

View larger version (20K):
[in this window]
[in a new window]

FIG. 5. Analysis of colocalization of D_H and V_H antisense signals with _µ0 and with each other. (A) Locations of RNA FISH probes: red, detected with Texas Red; green, detected with FITC; blue, detected with Alexa 350. Three-color RNA FISH detection of D_H antisense or V_H antisense, _µ0, and I_µ transcription. (B) Locations of RNA FISH probes: red, detected with Texas Red; green, detected with FITC; blue, detected with Alexa 350. Three-color RNA FISH detection of D_H antisense, V_H antisense, and I_µ sense transcription. All experiments were performed with day (D)15.5 fetal liver B lymphocytes; representative examples of the different signals observed are given in each case. (C) Analysis of colocalization frequencies of D_H antisense or V_H antisense with _µ0 and I_µ. (D) Analysis of colocalization frequencies of D_H antisense, V_H antisense, and I_µ.

In contrast, V_H antisense, _µ0, and I_µ colocalizedin only 9% of total alleles, while V_H antisense transcriptswere detected without _µ0 on 32% of total alleles (Fig.5C). Thus, only 20% of V_H antisense signals were associatedwith _µ0. Together, these results suggest that D_H antisensetranscription precedes D_H-to-J_H recombination of the Igh locusin fetal liver, and that V_H antisense transcription can initiatebefore D_H-to-J_H recombination on a small number of alleles,but usually occurs concomitant with or following D_H-to-J_H recombination,as previously shown in BM (8).

Given the differing associations with germ line alleles, wehypothesized that D_H and V_H antisense transcription would rarelybe found on the same allele. To test this, we cohybridized probesto I_µ, D_H (DSP and DFL), and V_H (J558) antisense transcriptsin day 15.5 fetal liver B lymphocytes (Fig. 5B). We detectedI_µ in blue, D_H antisense in green, and V_H antisense inred (Fig. 5B). We found that D_H and V_H antisense signals colocalizedin 11% of alleles (Fig. 5D). Thus, while D_H and V_H antisensetranscription can occur simultaneously on a low number of alleles,the majority (89%; 55% D_H, 34% V_H) occurs separately. Thesedata demonstrate a developmental shift from D_H antisense transcriptionto V_H antisense transcription concomitant with D_H-to-J_H recombinationin fetal liver.

The Igh intronic enhancer E_µ regulates D antisense intergenic transcription. The data thus far provided strong correlative evidence in supportof a role for D_H antisense intergenic transcription in chromatinaccessibility for D_H-to-J_H recombination. To test this modelfunctionally, we assessed the role of the known regulatory elementsPDQ52 and the intronic enhancer E_µ in regulating D_H antisensetranscription.

The PDQ52 promoter region initiates _µ0 transcription inthe sense direction through the J-Cµ region and is alsothought to have enhancer function (33). We first asked if, similarto one other immunoglobulin promoter (45), PDQ52 was bidirectionaland thus also the promoter of the D_H antisense transcription.To test this, we designed reverse transcription and nested PCRprimers to amplify regions upstream and downstream of PDQ52and did reverse transcription-PCR on CD19⁺ Rag1^–/–BM RNA (Fig. 6). Antisense-specific products of similar bandintensities were amplified for both of these regions, indicatingthat antisense transcription occurs both upstream and downstreamof PDQ52 and, hence, PDQ52 is unlikely to be the promoter of,or to regulate, the antisense transcription. This suggestedthat the downstream enhancer E_µ might regulate D_H antisensetranscription. This was an attractive hypothesis, as we hadpreviously shown that E_µ was required for D-to-J recombination(2).

View larger version (29K):
[in this window]
[in a new window]

FIG. 6. Analysis of transcription surrounding PDQ52 in Igh WT Rag1^–/– mice. Reverse transcription sense and antisense primers were designed in regions 5' and 3' of PDQ52. Nested primers were then used in PCR. Sense transcripts corresponding to _µ0 were not detected due to placement of the 5' PCR primer upstream of the _µ0 transcription start site.

To investigate this hypothesis, we analyzed D_H antisense transcriptionin two gene-targeted mouse models we have previously generated(2). The first contains a 300-bp deletion of the region upstreamof DQ52 containing the _µ0 promoter PDQ52 and has no effecton V(D)J recombination of the Igh locus. The second containsa deletion of both PDQ52 and a 700-bp region spanning the coreE_µ and its 5' matrix attachment region (MAR). These micehave a dramatic defect in D_H-to-J_H recombination, which, inthe absence of a phenotype with the PDQ52 deletion alone, indicatesthat this process is regulated by E_µ alone (2, 49).

We examined D_H region sense and antisense transcription by reversetranscription-PCR in B220⁺ BM B lymphocytes from mice harboringthese deletions on a Rag-deficient background, which ensuredno interference from DJ_H- or VDJ_H-rearranged transcripts. D_Hantisense transcripts were detected in PDQ52^–/–mice at levels similar to WT (Fig. 7A), indicating that thisdeletion has no effect on D_H antisense transcription and supportingour reverse transcription-PCR data (Fig. 6). In striking contrast,the additional absence of E_µ caused a dramatic reductionin antisense transcription over both DFL and DSP gene segments(Fig. 7A and B). These data indicate that D_H antisense transcriptionis regulated, either directly or indirectly, by E_µ, whichalso regulates D-to-J recombination. In contrast, deletion ofE_µ did not inhibit V_H antisense transcription, demonstratingthat this process is not regulated by E_µ and correlatingwith the lack of effect of E_µ on V-to-DJ recombination(Fig. 7B).

View larger version (46K):
[in this window]
[in a new window]

FIG. 7. Reverse transcription-PCR analysis of D and V region germ line transcription in Igh WT, PDQ52^–/–, and PDQ52^–/–E_µ^–/– Rag1^–/– mice. (A) Design of the D_H gene germ line transcription reverse transcription-PCR assay. (B) D_H gene strand-specific reverse transcription-PCR of total RNA from B220⁺ BM B lymphocytes from Igh WT, PDQ52^–/–, and PDQ52^–/–E_µ^–/– Rag1^–/– mice. (C) Semiquantitative reverse transcription-PCR of D_H and V_H (V_HJ558) germ line transcription in WT and PDQ52^–/–E_µ^–/– Rag1^–/– mice. ${lambda}$ 5 was used as a loading control.

D_H antisense transcription initiates either within or immediately upstream of the E_µ core element. As we have previously shown that antisense transcription doesnot occur in the region immediately downstream of E_µ (8),we reasoned that the promoter of D_H antisense transcriptionwas located within E_µ or its immediate upstream flankingsequences and thus sought to map transcription further and identifyputative promoters in the region between DQ52 and E_µ.Bioinformatic analyses using Genomatix Promoter Inspector (10)did not identify any canonical promoter elements. We attemptedstrand-specific reverse transcription-PCR across this intervalusing RNA from Igh-WT Rag-deficient mice. However, this provedimpossible due to detection of the high-level _µ0 sensetranscription in the antisense-specific and primer-negativecontrols due to endogenous priming in the reverse transcriptionstep (not shown). We considered the possibility that _µ0primary transcription, and hence endogenous priming, might besignificantly reduced in the PDQ52^–/– Rag-deficientmice and that these mice could thereby be used to map the antisensetranscription within this region. We have previously shown thatlevels of mature, spliced _µ0 mRNA are little changed whenPDQ52 is deleted (2) but had not assessed _µ0 primary transcriptionin these mice. Semiquantitative reverse transcription-PCR usingprimers in the _µ0 intron demonstrated that _µ0 primarytranscription is six- to eightfold reduced when PDQ52 is deleted(data not shown).

As it seemed likely that the antisense transcription was initiatingeither within or immediately upstream of E_µ, we analyzedtranscription between J4 and the 222-bp E_µ core (22) inPDQ52^–/– mice. We did strand-specific reverse transcription-PCRusing reverse transcription primers located immediately 3' ofJ4 (antisense specific) and at the 3' margin of the E_µcore (sense specific), followed by PCR using three sets of primersin the intervening region (Fig. 8). Antisense-specific productswere amplified for the two 5' regions but not the 3' regionspanning the E_µ core, demonstrating that transcriptioninitiates either within the E_µ core or in the 235-bp regionimmediately upstream.

View larger version (45K):
[in this window]
[in a new window]

FIG. 8. Mapping of an antisense transcriptional start site to the 5' margin of the E_µ core. Reverse transcription primers were designed immediately 3' of J4 and at the 3' boundary of the E_µ core to detect antisense and sense transcription, respectively, across this interval. Three sets of PCR primers in the intervening region were then used to map transcription across this region. Above lanes, with (+) or without (–) reverse transcriptase (RT). DNA, genomic DNA positive control.

DISCUSSION

Top

DISCUSSION

We report the discovery of antisense intergenic transcriptionthroughout the D_H and J_H regions of the mouse Igh locus in pro-Bcells and propose that this process remodels the D_H clusterfor D_H-to-J_H recombination. In support of this model, we showthat D antisense transcription is activated before D_H-to-J_Hrecombination and is regulated by the intronic enhancer E_µ.E_µ was recently demonstrated to regulate V(D)J recombinationof the Igh locus at the earliest D_H-to-J_H recombination step(2). Taken together, these data suggest that E_µ controlsD_H-to-J_H recombination by activating this form of germ lineIgh transcription and that the processivity of this transcriptionrenders the D region accessible for D_H-to-J_H recombination.To our knowledge, this is the first report for an endogenouslocus of a regulatory element (E_µ) regulating both intergenictranscription and a process that requires chromatin accessibilityof the transcribed region, in this case, D_H-to-J_H recombination.This model is supported by a recent report that intergenic transcriptionregulates J ${alpha}$ gene recombination in the TCR ${alpha}$ locus (1).

In contrast to transcription over the D region, we show thatdeletion of E_µ has no effect on V_H antisense transcription,suggesting that accessibility of the V_H region is not regulatedby E_µ, in agreement with E_µ's not having a majorrole in V_H-to-DJ_H recombination (2, 49). Furthermore, we showthat D_H antisense transcripts initiate on germ line allelesand that D_H and V_H antisense transcripts are rarely associatedon individual alleles. Thus, we demonstrate a stepwise progressionof antisense intergenic transcription, first over the DJ region,under the control of E_µ, then over the V region, underthe control of a separate, unknown, regulatory element. In bothcases, transcription precedes the corresponding V(D)J recombinationevent. This pattern is strikingly similar to the observed stepwiseprogression of active histone marks during Igh V(D)J recombination(15). Our results raise an important question: what regulatesV region intergenic transcription? We have ruled out the PDQ52promoter/enhancer, since V antisense transcription is normalin PDQ52 knockout mice. Interestingly, a novel pro-B cell-specifichypersensitive site has recently been identified 5' of the Vregion, although its function is not yet known (48).

We have discovered that a major transcription start site forD region antisense transcription lies either within the E_µcore or in the upstream J-proximal 235-bp region including the5' MAR (Fig. 8). Since the enhancer core and upstream regionare both deleted in the E_µ knockout mice we analyzed here,we are currently unable to distinguish between a putative antisensepromoter and the known enhancer functions of the E_µ core.However, we have compared these mice with mice that lack theE_µ core but have the upstream region intact (49), andboth have a similar defect in Igh D-to-J recombination (2).This suggests a dominant role for the E_µ core in thisprocess, either as a bona fide enhancer, an as-yet-unclassifiedpromoter, or both.

We propose that antisense intergenic transcription over D_H facilitatesthe establishment of an active chromatin domain for D_H-to-J_Hrecombination, either by histone exchange or recruitment ofchromatin modifiers. Our model is supported by several observations.First, intergenic transcription is concomitant with a decreasein repressive histone marks, including dimethyl histone H3K9,and an increase in active histone marks, including H3 acetylation,and chromatin-remodeling enzymes over the DJ_H region prior toD_H-to-J_H recombination (15, 37, 43). Histone acetylation ishighest over the J region but is also widespread throughoutthe D region (15). This is consistent with histone acetylation'sbeing propagated by intergenic transcription initiating nearE_µ and decreasing with increasing distance from the startsite. However, it is not yet clear whether there is a continuousantisense transcript starting close to E_µ or multiplesmaller transcripts initiating throughout the D locus. Sincethis is a large region (60 kb), it is also possible that E_µcontrols transcription in cooperation with more than one antisensepromoter. This model is in agreement with a recent suggestionthat the region encompassing DQ52 and the four J genes and E_µforms a separate chromatin domain from the rest of the D region(37).

Conversely, this study provides substantial evidence opposinga role for antisense transcription in generation of double-strandedRNA (dsRNA) and heterochromatin formation in the Igh locus.Strikingly, the J region, which displays the highest histoneacetylation before D-to-J recombination, forms part of the transcriptionoverlap between the _µ0 sense transcript and antisensetranscription originating near E_µ, suggesting that thetranscription units are not antagonistic. Indeed, they are coordinatelyupregulated by E_µ. Furthermore, over DQ52, the singleD gene that expresses sense transcripts, D_H antisense and _µ0(sense) are cotranscribed on the same allele, yet DQ52 is rathermore acetylated than the rest of the D genes (15, 43) and ispreferentially used in early D-to-J recombination (6). Additionally,our finding that there is no sense germ line transcription inthe remainder of the D_H region indicates that there is no possibilityfor dsRNA formation. Intergenic transcription has recently beenshown to be required for V(D)J recombination at the TCR ${alpha}$ locus,but in this case, it originates from the sense strand (1). Thissuggests that the strand origin is not important, which supportsour model that the processing activity is the key function ofthis transcription.

Why is a large-scale chromatin remodeling process necessary?The D_H genes are small (11 to 16 bp) relative to the large intergenicsequences between them (4 kb). However, relocation of the Ighlocus from the nuclear periphery to the interior precedes D-to-Jrecombination and requires chromatin unfolding (32). Furthermore,Igh must be made accessible to the large RAG recombinase complex.It is unlikely that either could be achieved through remodelingof the genes alone. Instead, intergenic transcription may alterthe chromatin structure throughout the large DJ region (60 kb)in a similar manner to other large developmentally regulatedloci (40), which undergo transcription-dependent (39, 44), higher-orderchromatin remodeling and looping out of their chromosome territories(13, 62). Furthermore, we speculate that E_µ may promotenuclear relocation by recruiting the D region to a transcriptionfactory in the nuclear interior to facilitate intergenic transcription,in a similar manner to the ß-globin locus controlregion (50).

Recombination of most antigen receptor loci (e.g., TCR ${alpha}$ , TCRß,and Ig ${kappa}$ loci) requires the cooperative activity of germ linepromoters and enhancers and is characterized by transcriptionfrom germ line promoters (34). Of particular interest are thepromoters upstream of each Dß gene cluster in theTCRß locus (PDß1 and PDß2) (56)and both upstream of and within the TCR ${alpha}$ J cluster (27), whichare required for efficient Dß-Jß (63, 64)and V ${alpha}$ -J ${alpha}$ (27) joining, respectively. The transcribed regionsare associated with active histone marks such as acetylation(41, 64). These promoters require the presence of their respectiveintronic enhancer elements (E_ß and E) for transcriptionalactivity (34). We have recently shown that E_ß hastwo distinct roles prior to V(D)J recombination of the TCRßlocus: an intrinsic function that directs general chromatinopening over most of the DßJß cluster anda cooperative function that facilitates the assembly of a promoter/enhancerholocomplex with PDß1 (46). Here we show that theD_H cluster differs from this and other antigen receptor lociin that transcription is undetectable from most D_H germ linepromoters. This raises the possibility that interaction betweenD promoters and E_µ may not occur and may not be necessaryfor D_H-to-J_H recombination. Further, deletion of the only promoterfrom which germ line transcription was detected, DQ52, had noeffect on antisense intergenic transcription, shown here, orD-to-J recombination (2), although in this case arguably a promoter-enhancerinteraction might not be necessary due to proximity of DQ52to E_µ. Thus, it is possible that E_µ enhancer-drivenantisense intergenic transcription may be the major force drivingaccessibility over the D_H region prior to DJ recombination.However, absence of transcription does not preclude interactionof the germ line promoters with E_µ, and such an interactionmay occur before recombination and expression of the D_µtranscript. It will be interesting to distinguish between thesepossibilities by direct analysis of the interaction of theseelements.

In summary, we have discovered that, in addition to the V_H region,antisense intergenic transcription occurs throughout the IghD region and is thus a widespread process during V(D)J recombination.D_H antisense intergenic transcription precedes D_H-to-J_H recombinationand initiates near to and is regulated by E_µ. Since E_µalso regulates D_H-to-J_H recombination, antisense intergenictranscription may provide a long-distance, processive mechanismby which E_µ is able to regulate accessibility across theD_H region.

ACKNOWLEDGMENTS

We thank Peter Fraser and Lyubomira Chakalova for critical reviewof the manuscript and Geoff Morgan for help with FACS.

D.B., A.W., and A.E.C. were supported by the Biotechnology andBiological Sciences Research Council including grant numberBB/C508769/1 (D.B.), and R.A. and E.M.O. were supported by grantsfrom the National Institutes of Health (P01 HL68744 and CA100905,E.M.O.; T32 CA09385, R.A.) and a Cancer Center Support grant(P30 CA68485, Vanderbilt-Ingram Cancer Center).

We declare that we have no financial interests that pose a conflictof interest with regard to this article.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Chromatin and Gene Expression, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom. Phone: 44 1223 496397. Fax: 44 1223 496022. E-mail: anne.corcoran{at}bbsrc.ac.uk

Published ahead of print on 25 May 2007.

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

REFERENCES

Top