Eur. J. Immunol. 1995. 25: 257-261 Human germ-line IgH chain transcripts initiate upstream of DQ52 257

Allan Thompson., Erik Timmers.., Ruud K. B. Schuurman. and Rudolf W. Hendriks.0

Department of Immunohaematologyo, University Hospital Leiden, Leiden, Department of Haematology., University of Cambridge, Cambridge and Department of Cell Biology0 and Genetics, Erasmus University, Rotterdam

Immunoglobulin heavy chain germ-line JH-C, transcription in human precursor B lymphocytes initiates in a unique region upstream of DQ52*

From human precursor B cells which had both immunoglobulin (Ig) heavy (H) chain loci in germ-line configuration ,various IgH chain germ-line transcripts were isolated and sequenced. These transcripts were shown to contain sequences derived from the JH region, the IgH chain enhancer element or the Ig switch region. A number of isolated cDNA clones contained sequences at their 5’ end that were derived from a single exon located just upstream of DQ52, designated the pO’element. Sequence analysis of a 920-bp genomic DNA segment, containing the po’exon and its 5’ flanking region, revealed the presence of various conserved motifs for DNA-binding proteins, such as E2A, Ets, NF-xB and AP-2,which have previously been found in the IgH and L chain enhancers. We propose that the activity of the po’ element, resulting in germ-line transcription of the DQ52-JH gene segment, is required to generate full accessibility for the V(D)J recombi- nase.

1 Introduction

The variable regions of immunoglobulin (Ig) and Tcell receptor (TcR) genes are assembled form germ-line V, (D), and J gene segments by an ordered series of-site-specific DNA rearrangements conferred by a common V(D)J recombinase [l-41. As this recombinase appears to be active throughout early B and T cell development [3-61, its activity must be regulated at the level of the template. It has been proposed that targeting of the recombinase to specific Tg or TcR segments is conferred by a specific increase of locus accessibility [5 , 61. Since Ig loci were found to be transcriptionally active at the time of rearrangement [S-ll], transcription might be a prerequisite for recombi- nation to occur. Alternatively transcription may only reflect locus accessibility, which is provided by the action of yet-unidentified factors.

In early stages of B cell development V(D)J recombination is initiated at the Ig heavy (H) chain locus with the juxtaposition of a D segment to a JH segment on both chromosomes. The IgH chain enhancer element (E,) is thought to play an important role in the initiation of VH-D-JH recornbination, as this element was shown to

[I 134871

* These studies were partially supported by the Dutch Praevention Foundation, the Royal Netherlands Academy of Arts and Sciences and the Dutch Foundation for Science.

Correspondence: Rudolf W. Hendriks, Department of Cell Biolo- gy and Genetics, Erasmus University Rotterdam, PO BOX 1738, NL-3000 DR Rotterdam, The Netherlands

Abbreviations: ASS: Acceptor splice site BLCL: B lympho-

induce both transcription and rearrangement in an artificial substrate introduced into murine B lymphocytes [12]. Recent studies have shown that other lymphoid or non- lymphoid enhancer elements can also target rearrangement of this substrate [13]. Deletion of the E, region from the mouse genome severely impaired but not completely eliminated VH-D-JH recombination [14, 151. Therefore, the existence of additional &-regulatory elements which, together with E,, are required to generate full recombina- tional accessibility, was suggested.

Her we report the identification of various IgH chain germ-line transcripts in human precursor B cells. Transcrip- tion appeared to initiate from a single exon located 0.5-1 kb upstream of DQ52.

2 Materials and methods

2.1 Cell lines and patients

The B lymphoblastoid cell lines (BLCL) YF.PB1-YEPB6, YEBM and RF.BM have been described before [16]. UNRO was established from peripheral blood from a pre-B acute lymphoblastic leukemia (ALL) patient (CD19+, CD10+, TdT+ , cytoplasmic p H chain positive). Leukemic cell samples were from patients that were classified as common-ALL (CDl9+, CD10+, TdT+, cytoplasmic p H chain negative).

The IgH and L chain gene configurations in cell lines and leukemic cell samples were evaluated using JH-, JK- and Ch-specific probes [ 161.

2.2 Construction and analysis of cDNA libraries blastoid cell line DSS: Donor splice site 3’ENH: IgH chain 3’ enhancer E,: IgH chain intron Enhancer S: Switch region TdT Terminal deoxynucleotidyl transferase

TheYF.PB2 and REBM oligo-(dT) primed cDNA libraries have been described [7]. Following essentially the same method, a third cDNA library (containing 3 x lo5 plaque-

Key words: B cells / Rearrangements / Germ-line transcripts forming units) was constructed from &.BM poly(A)-

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1995 0014-2980/95/0101-0257$10.00 + .25/0

258 A . Thompson. E . Timmers, R. K. B. Schuurman and R. W. Hendriks Eur. J. Immunol. 1995. 25: 257-261

enriched RNA using the C,-specific primer IGHCMU. Screening of the cDNA libraries was carried out according to standard procedures [ 171 using GeneScreenPlus hybrid- ization membranes (Dupont Company, Boston, MA). The probes applied, a human IgH C, probe (1.3-kb EcoRI fragment [ 18]), a human po' probe (650-bp PvuII fragment, see Fig. 3A), a human JH region probe (2.5-kb EcoRI-BglII fragment [18]) and a human switch region S, (4.3-kb BglII-Dsal fragment [18]) probe, were radiolabeled by random priming (Boehringer Mannheim).

2.3 DNA subcloning and sequencing

Inserts from cDNA libraries were subcloned into M13 vectors by PCR amplification using hgtl0 primers that contained restriction sites. PCR products generated by amplification of first strand cDNA were purified and cloned into pIC20H, as previously described [19].The 650bp PvuII fragment upstream of the q J ~ l gene segment was isolated from a human genomic BamHI-Hind111 clone encompass- ing the JH and E, region (kindly provided by m. Alt, Boston, MA) and subcloned in the SmaI site of pBluescript KSII- (Stratagene).

DNA sequencing was by dideoxy chain termination method [17], using M13 forward and reverse primers and internal primers designed on the basis of the obtained sequence. The UWGCG sequence analysis software pack- age version 7.1 was used. Homology search was performed using the FASTA program in the EMBL and GenBank data bases.

2.4 Blotting procedures, reverse transcription (RT)-PCR and probes

Isolation of total cellular RNA and Northern blotting analyses were performed as described [7]. For RT-PCR, aliquots of 2-5 pg total cellular RNA were used for first strand cDNA synthesis with M-MuLV RT (Pharmacia P-L Biochemicals), using the IgH chain C,-specific primer IGH- CMU 5'-TGCATGACGTCCTTGG-3'. Wirth first-strand cDNA as templates, PCR products were generated in 40 cycles of amplification, using primer IGHCMU in combi- nation with a po'-specific primer [IGHMUO:-5'-CACCAT- GGGAAGAGCTGA-3'; (see Fig. 3A; pos. SSS)] or a JH1-specific primer (IGHJ1: 5'-GGACACAGTGATTGG- CAG-3'). PCR conditions and Southern blotting analyses of amplified products have been described previously ~191.

2.5 RNase protection analyses

Complementary-strand RNA probes were synthesized by in vitro transcription (Stratagene, La Jolla, CA) from a p' (650-bp PvuII fragment) or a no template (325-bp KpnI- ClaI) in pBluescript KSII- (Stratagene), using T3 or T7 polymerase and [32P] UTF! In RNA protection assays 20 pg of total RNA was hybridized overnight at 30, 45 or 60°C with the radiolabeled complementary strand RNA probes in 30 p1 of hybridization buffer (Boehringer Mannheim). After digestion with RNase A and T1 at 30°C for 30 min, the protected fragments were ethanol precipitated and analyzed on a 6 % polyacrylamide gel, with previously analyzed po' sequencing reactions as size markers.

cP E N s w

R 1 2 9 4

7 5 5 \ . DNA

H 1 Kb

GT.JP

GT.J3

Figurel. Overview of the IgH chain cDNA clones isolated from human pre- cursor BLCL. A map of the IgH chain locus is drawn to scale. The pa' region is represented by an open box, while enhancer (EN) and switch exons (SW) are indicated by hatched boxes.The four categories of cDNA clones are shown, as alligned to the position of their JH segment region -if present - on the IgH chain map.

-&-~~:~:~:~:~>]

Eur. J. Immunol. 1995. 2.5: 257-261 Human germ-line IgH chain transcripts initiate upstream of DQ52 259

3 Results

3.1 IgH chain germ-line transcription in early precursor B cells

Expression of IgH chain germ-line transcripts was analyzed in EBV-transformed BLCL derived from bone marrow or peripheral blood from two patients (YF and RF) with a novel type of SCID [16]. These lines reflect the earliest detectable stage in human B cell differentiation in that they express B cell-specific membrane determinants, but still have both their IgH and loci in the germ-line configuration. In a Northern blot analysis C,-specific transcripts were detected (data not shown). To characterize these IgH chain germ-line transcripts cDNA libraries were constructed and C,-positive inserts were isolated and sequenced. On the basis of their structure the characterized cDNA clones were classified into four distinct families (Fig. 1).

The first group of cDNA contained, next to Cp, only one or multiple JH elements. Two different GT. J2 cDNA clones, which were isolated from an oligo(dT)-primed cDNA library, were found to be either of the y membrane or the y secreted form.

The second group of cDNA clones contained - mostly in addition to (multiple) JH segments - a unique 320-bp E, exon spliced to JH6 or JH1 (see Fig. 1; GT.J6.EN and GT.J1 .EN). This E, exon was localized 460-780 bp down- stream of the E, octanucleotide, uncovering a novel acceptor splice site (ASS; Fig. 2). The donor splice site (DSS) utilized was the same as the one previously described to be present in I, transcripts, which initiate within the human E, element just downstream the octamer sequence 1111.

Within the third category, one cDNA (GT.J3.SW) con- tained sequences derived from J"3 and its upstream IVS, as well as a novel 200-bp exon derived from a region localized 500-700 bp upstream of C, (Fig. 2). The identified ASS is just downstream of the pentameric repeat region of S, [20].

ASS DSS I

sw DNh < C*TO*TCTTG Ti00iiCliT*C ATcoIITpiucc TccIIccmAGT I / CCTCCliCCCT TC*C&TTCAG CTAmCAAc Crn'%CCCTCCc >

W . J 3 sw < ArnCTCCltCCD G T C X T X I C - . / / . . ..

J 3 DNA < AmCCTCIICCG CXTCTTCAO.

MU DNA .CC*GTGC*TC ccccccIvicc >

GGROlOCATC coccccAAcc >

Figurr 2. Genomic introdexon boundaries, showing the ASS and DSS of enhancer exon (upper half) and switch exon (lower half). The cDNA clones GT.Jl.EN, GT.J6.EN, and GT.J3.SW are alligned to the genomic sequences of the human E,, JH, C, and S, regions. A closed triangle indicates where the germ-line sequence would have continued had splicing not occurred. Homology is depicted by a dash. A double slash depicts internal sequences of the enhancer and switch exons not shown.

Other cDNA clones (GT.SW) appeared to run through the ASS and DSS sites and extended up to 2 kb upstream of C,, well into the highly repetitive region of S,.

3.2 IgH chain germ-line transcripts containing the po' element

The fourth group of cDNA clones contained 5' sequences which did not show significant homology to any human sequence in the DNA database, immediately followed by a 61-bp segment identical to the 5' end of the PvuII genomic fragment that contains the human D Q ~ ~ - J H region [18]. As this finding suggested that the cDNA clones contained sequences immediately upstream of this PvuII fragment, we determined the sequence of 920 bp of 5' flanking genomic DNA, extending to a BamHI site (Fig. 3).

Comparison of the genomic sequence with the character- ized cDNA clones revealed that the 5' end of the clones GT.MuO and GT.MuO.Jl were derived from a single exon. This exon was designated po', analogous to the yo transcripts which initiate 38-87 upstream of DQ52 in the mouse [9, 10,211. The po' element was generally found to be spliced to the first C, exon (see Fig. 2; GT.Mu0 trans- cripts), using a DSS 437 bp upstream of c p J ~ l (Fig. 3A). One cDNA, GT.MuO.J1, was found to run through this DSS and contained cpJ~1, DQ52 and J H ~ elements. The majority of po'-containing cDNA clones had 5' ends located 485 to 605 bp upstream of the c p J ~ l segment (Fig. 3A).

In RNase protection assays using RNA derived from YF and RF BLCL, multiple protected fragments were observed that were localized within the same region as defined by the 5' end of the sequenced cDNA clones (Shown for REBM in Fig. 3B). Apparently, there is heterogeneity of the yo' transcription start site with two main regions of initiation, within positions 710-750 bp and 830-860 bp respectively (Fig. 3). This heterogeneity is in contrast to the single start site observed for germ-line xo transcription [7] from the Ig x locus in the YF and RF cell lines (shown for RF.BM in Fig. 3B).

Due to the presence of termination codons in all three reading frames of the p' region, there was no significant open reading frame in any of the yo'-containing cDNA.

3.3 Identification of motifs for DNA binding proteins in the po' element

The po' element contains various conserved motifs (Fig. 3A). About 600 bp upstream of the region of trans- cription initiation, three E2A consensus sequences [22] were identified, one of which was located within a box (position 57-64) identical to pE5 in the mousehat 3' ENH [22-241. A fourth E2A consensus was present at position 1043. A sequence identical to the pE4 box in the mouse E, was found at position 544-551. Three motifs for ets-domain proteins were identified, two of which were close to the E2A boxes (at positions 340 and 538) and one was located further downstream (at position 1095). Furthermore, the po' element contains five AP-2 consensus sequences and an NF-xB site (positions 849-858).

260 A. Thompson, E. Timmers, R. K. B. Schuurman and R. W. Hendriks

B-1 GGATCCIWC occacc_ccrc TDICACCTCCT GTCCTTTCCC TGGTOTCTGC TTOT

Figure 3. (A) Genomic sequence of the po' region (EMBL accession number X80347). The restriction sites for BamHI and PvuII are indicated. Asterisks show 5' ends of po' cDNA clones isolated. Open triangles denote the position of transcription start sites determined by RNase protection assays. A vertical arrow shows the DSS, where splicing of the po' exon to C, occurs. AP-2 binding sites are doubly underlined (consensus = T/CC/GC/GCCA/CNG/cC/GG/c or GGGA/GHT%T/cCC). E2-A and pE4 boxes are shaded (consensus = "[ACAGNTG and CAGGTGGT, respectively).The position of the NF-KB binding site is indicated by a dotted line (consensus = GGGA/GH7?/cT/cCC). H = A, Tor C. The Ets binding motifs are singly underlined (consensus C/AGGAA/T).The DNA sequence of position 921-1430 has been published previously [ 181 (EMBL accession number J00256). (B) FWase protection assay showing transcription initiation sites in the human Ig po' (lanes 1-6) and Ig no region (lanes 7-12). The non-hybridized undigested complementary sense riboprobes are shown in lane 1 (generated from a cloned 650 bp PvuIl pa' fragment (Fig. 3) and in lane 7 (generated from a 375 bp ClaI-Kpn xo fragment [7]). Hybridizations were at 45 "C with either 20 pg total RNA (lanes 2 and 8) or 2 pg polyA(+) RNA from BLCL RF.BM (lanes 3 and 9). Further hybridizations were with 20 pg total RNA from RF.BM at 30 "C (lanes 4 and lo), or at 60°C (lanes 5 and 11). Control hybridizations were performed with 20 kg of tRNA at 45 "C (lanes 6 and 12). Previously analyzed po' sequencing reactions were used as size markers, indicated in bp.

Eur. J. Immunol. 1995. 25: 257-261

B 1 2 3 4 5 6 789101112

-660

-375

-300

-250

-150

-100

3.4 pn' expression in precursor B cells

Germ-line IgH chain transcripts were not only present in precursor BLCL derived from the SCID patients, but also in three peripheral blood cell samples from CALL patients and a pre-B cell line UNRO derived from a pre-B ALL patient.The analyzed cells had one germ-line IgH allele and one rearranged (either p+ or p-) allele. In RT-PCR experiments using either a p"'-specific primer or a primer just upstream of JH1 together with a Cp-specific primer, in all four samples products were amplified which hybridized to a Cp probe (data not shown).

4 Discussion

We have characterized the structure and processing of various human IgH chain germ-line transcripts. Such transcripts were present in precursor B cells which had either both H chain loci in the germ-line configuration, or one germ-line allele and one - functionally or non- functionally - rearranged allele.

Using RNase protection assays we observed that germ-line transcripts initiated with extensive 5' heterogeneity within a region located about 0.5 kb upstream of pseudo-JH1, designated p"'. The heterogeneity of transcription initiation appears to be consistent with the absence of an octamer (which is present in 3t0 [13]) or TATA box in the pO'element.

It remains unclear whether the identified cDNA clones that do not contain this po' region all reflect fragments of a single heterogeneously processed large transcript that initiates in p"' and runs through the JH, E,, S, and C, regions, or alternatively, arise from several different transcription initiation sites further downstream within the JH-C, inter- val. The latter may perhaps only be the case for those transcripts (GT.EN, Fig. 2), that were found to be similar to the I, transcripts, [11].

The IgH chain germ-line transcripts in human are different from those described in the mouse, as in the mouse these transcripts initiate 38-87 bp upstream of DQ52 and run through the JH and C, gene segments [9, 10,211. In the mouse, the DQ52 5' flanking region was shown to exhibit interactions with several transacting factors in a tissue- restricted manner and to have both promoter and enhancer activity, mostly confined to an area 61-145 bp upstream of DQ52 [21, 251 containing two Ap-1-binding sites. There- fore, this DQ52 flanking region may function as the murine counterpart of the human p"' region. We were able to identify consensus sequences for E2A, NFxB, Ets and AP-2 domains, each of which are also present in the IgH and L chain enhancers. Especially the presence of E2A-boxes in the po' element may be important, as the E2A-binding helix-loop-helix protein E47 has been implicated in the regulation of IgH chain rearrangement [26, 271. However, the significance of the observed homologies still has to be demonstrated in functional studies.

Eur. J. Immunol. 1995. 25: 257-261 Human germ-line IgH chain transcripts initiate upstream of DQ52 261

Early in B cell differentiation, activity of the po’ element will lead to transcription of the DQ52 and J H region, and subsequently into D-JH recombination. The unique loca- tion of the DQ52 element downstream of po’ may account for its preferential usage in primary D-to-JH recombina- tions. The recent finding that D-to-J, recombination is not blocked by targeted deletion of E, from the mouse genome [14] would be consistent with the D-to-JH recombination process being mainly under control of the po’ element. As the pO’element will be deleted in all D-to-JH recombination events except those in which DQ52 is involved,V-to-DJH recombination should be under the control of E,. This is consistent with the finding that V-to-DJ, recombination is severely impaired in E, knock-out mice [14, 151. We conclude that our findings in human precursor B cells support the conclusion by Kottmann et al. [21] based on findings in the mouse, that the order of IgH chain rearrangements, first D to JH, then V to DJH, could be explained by the specific interplay between the po’ and E, elements.

We thank E: Smier~s (Dept. Haematologx Erasmus University Rotterdam) for detailed analyses of samples from A L L patients, R . Offringa and P van den Elsen (Dept. Immunohaematology, University Hospital Leiden) for critical reading of the manu- script.

Rcceived September 14, 1994; in revised form November 5, 1994; accepted November 8, 1994.

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