MOLECULAR AND CELLULAR BIOLOGY, OCt. 1990, p. 5340-5348 0270-7306/90/105340-09$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 10

Parameters That Govern the Regulation of Immunoglobulin 8 Heavy-Chain Gene Expression ROLAND TISCH, NAOMI KONDO,t

AND

NOBUMICHI HOZUMI*

Mount Sinai Hospital Research Institute and Departments of Immunology and Medical Genetics, University of Toronto, 600 University Avenue, Toronto, Ontario, Canada MSG IX5 Received 11 May 1990/Accepted 26 July 1990

The ,u and 8 immunoglobulin heavy-chain genes comprise a complex transcriptional unit in which a single mRNA precursor gives rise to ,u- and 8-specific transcripts. During the immature B-cell stage, posttranscriptional processing events involving alternate splicing and cleavage-polyadenylation site selection give rise to Jibut not 8-encoding transcripts. In terminally differentiated B cells, 8 mRNA is not synthesized because of a transcription termination event occurring upstream of the 8-gene locus. In an attempt to gain insight into the respective contributions of alternate splicing and cleavage-polyadenylation in the control of 8 mRNA synthesis, we have constructed a set of plasmids in which membrane ,L (l.m)-B intergenic sequences containing the ILm poly(A) site but differing in splicing capacity were inserted in between a VH and 8 gene. The .m4-8 insertion vectors were transfected into a B lymphoma line representative of an immature stage, and proximal ILm poly(A) site usage and 8 mRNA synthesis were assessed. To determine unequivocally whether the p1,-8 intergenic region can regulate termination, the insertion vectors were also transfected into a B myeloma line, and transcription through the region was measured. In immature B-cell transfectants, splicing site selection was found to have a key role in determining poly(A) site utilization and concomitant 8 mRNA expression. Mature 8 mRNA synthesis was blocked by an upstream cleavage-polyadenylation event only when the proximal poly(A) site was associated with appropriate splicing signals. Furthermore, in vitro transcription assays revealed that the iLm-8 intergenic region is sufficient to regulate transcription termination within a 1,243-base-pair region containing the Lm poly(A) site in myeloma transfectants. The Lm-t8 insertion vectors provide an excellent model system for studying the regulatory aspects of this transcription termination event. usage, where selection of a specific poly(A) site would determine the appropriate overall RNA splicing pattern (7, 32). Alternately, a mechanism in which active regulation of splicing events may be utilized. The selection of ,u- or 8-associated splice sites in a developmental fashion could determine the availability of specific poly(A) sites. Such a mechanism is believed to be utilized in the differential expression of calcitonin/CGRP mRNA (14). Little is known about the regulatory aspects of the transcription termination event that occurs within the p.-8 unit in terminally differentiated IgM-secreting B cells. Nucleotide sequences acting as putative termination signals or involved in establishing DNA or RNA secondary structures necessary for the termination process still require functional elucidation. Furthermore, there are confficting data regarding the precise site of transcription termination, which has been mapped to both Vus-Lm (7, 9) and .m-8 (10, 13, 18, 32) intron regions. Localization of the termination site would aid in elucidating the requirements for this process. The experiments reported here were designed to (i) examine the factors that can influence the differential synthesis of 8 mRNA in early B cells and (ii) determine unambiguously whether the p.m8 intron region can direct transcription termination in terminally differentiated B cells and, if so, identify sequence requirements for the process. The approach taken makes use of a number of recombinant DNA constructs in which various DNA segments found between .M- and 8-encoding sequences were inserted upstream of a 8 heavy-chain gene. After transfection into B cell lines representing distinct stages in B-cell maturation, these constructs were assessed for 8-gene expression and associated poly(A) site utilization or transcription termination. We report here that splice site selection appears to have a key role in

Complex transcriptional units encode multiple mature mRNAs generated from a common RNA precursor (15). One such example is the p.-8 immunoglobulin heavy-chain transcriptional unit expressed in B lymphocytes (Fig. 1). The ,u and 8 heavy-chain genes share the same transcriptional promoter (11), and both utilize the identical upstream variable heavy-region gene segment (VH) (16). The production of distinct ,u and 8 mRNAs is achieved in a B-cell stage-specific manner by alternative posttranscriptional processing of a ix-8 primary transcript involving cleavage and polyadenylation [poly(A)] and splice site selection and by differential transcription termination (18, 32). Immature B cells synthesize mRNAs encoding the membrane (Rum) and secreted (R,.) forms of the ,. heavy chain, whereas 8 mRNA is not expressed. Despite this lack of mature 8 mRNA, expression studies of the [.-8 unit have demonstrated that the 8 gene is transcribed. In mature B cells, mRNA encoding for the 8 heavy chain is detected in addition to p,s and p.m transcripts. Upon differentiation of a mature B cell into an immunoglobulin M (IgM)-secreting plasma cell, R.u mRNA becomes the predominant species and 8 mRNA is essentially undetectable. This marked reduction in 8 mRNA expression is chiefly due to transcription termination occurring upstream of the 8 gene.

The details of the respective contributions of alternate poly(A) and splice site selection in the developmental control of 8 mRNA synthesis are unclear. Regulation of 8 mRNA synthesis may reside at the level of poly(A) site

Corresponding author. t Present address: Department of Pediatrics, Faculty of Medicine, Gifu University, Gifu, Japan. *

5340

IMMUNOGLOBULIN 8 HEAVY-CHAIN GENE EXPRESSION

VOL. 10, 1990

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determining poly(A) site utilization and that the Um-5 intron region alone is sufficient to regulate appropriate transcription termination. MATERIALS AND METHODS Plasmid constructions. The vector pRCbs was constructed to facilitate insertion of the U intron region segments used in this study. In brief, a rearranged 8TNp-chain gene was inserted into the vector pSV2-neo, which confers resistance to the antibiotic G418. A unique Sall site was generated in place of an EcoRI site found between the VHTNP and b-chain genes. This SalI site was used for subsequent subcloning of the lLmb inserts. The 5' and 3' ends of each insert were converted into Sall cohesive ends by Klenow end filling and Sall linker ligation. The p.~5 insert is a 2.5-kilobase (kb) KpnI-EcoRI fragment containing the p., exons and associated PUm poly(A) site. BAL 31 exonuclease digestion was used to construct the pBRu..b, M28, and u..30+8 inserts. The precise ends generated by this digestion were determined by DNA sequencing. Starting from the KpnI site, 650 and 732 base pairs (bp) were removed in the pBRp.mj and M28 inserts, respectively. The pBRjijb insert retains 67 bp of the Ml exon, whereas the Ml exon is completely removed in the M28 insert. A 464-bp EcoRV-Sall pBR322 fragment was used to replace sequences deleted in the pBRp.mb insert. All but 30 bp downstream of the p.m cleavage site was removed in the R,m30+8 insert. The R,mTK8558 insert was constructed by replacing a 870-bp XhoI-EcoRI fragment with a 855-bp BamHI-HindIII noncoding segment found upstream of a CK light-chain gene. To construct the Ap.LJ insert, a 900-bp KpnI-XbaI fragment containing the P,u exons was removed, leaving the pi.. poly(A) site and downstream sequences intact. The 3'SP5 insert was generated by ligating a 200-bp HindIII-HpaI fragment from a CK lightchain gene to a Sall site (formerly an XbaI site) found 116 bp upstream of the p., poly(A) site. This fragment contains the 3' splice acceptor site and 9 bp of CK-encoding sequence in addition to 200 bp of upstream intron sequences. A 255-bp SaII-BamHI fragment containing the simian virus 40 (SV40) early-gene poly(A) site was isolated from the pECE vector (5) and used for the p,.SV408 insert. A 900-bp KpnI-XbaI p.m exon-containing segment or the CK 3' splice acceptor-carry-

ing fragment was placed upstream of the SV40 poly(A) site for the p.mSV408 or 3'SPSV408 insert, respectively. The pRp.-8 vector was constructed by subcloning a 12.2-kb partial EcoRI fragment containing the 8 heavy-chain gene into the PqTNP gene-carrying pR-Sp6 construct (22). Cell lines and DNA transfection. The B lymphoma M12.4 (12) and plasmacytoma J558L (23) were used as gene transfer recipients. Electroporation with a Bio-Rad Gene Pulsor was the method of choice to establish stable transfectants expressing the pLm-8 insertion constructs. G418-resistant clones were initially screened for expression of VHTNP-specific mRNA as determined by RNA dot blotting. Between 30 to 40 VHTNP mRNA-expressing clones were pooled for each set of transfectants and used for subsequent analysis. RNA preparation and analysis. Total cytoplasmic RNA was extracted and passed over oligo(dT) columns to isolate poly(A)+ RNA (28). RNA was fractionated on 1% agarose gels [5 p.g of poly(A)+] and transferred to Dupont GeneScreen Plus membranes. Hybridization with the appropriate radiolabeled DNA probes was performed as described previously (29). The VHTNP probe used is a 910-bp genomic fragment encoding 2,4,6-trinitrophenol (TNP) specificity; the neo probe is a 1.3-kb BgllI-HpaI genomic fragment isolated from pSV2-neo, and the 8 probe is a 861-bp PstI cDNA fragment isolated from p554j (20). For S1 nuclease analysis of the pm-8 insertion transfectants, total cytoplasmic RNA (30 p.g) or nuclear RNA (30 pLg) was hybridized to an excess of end-labeled double-stranded DNA probes. Two p.m poly(A) site-specific probes were used to analyze transfectant mRNA. The first probe is a 173-bp XbaI-SalI fragment in which 57 bp remains downstream of the pm cleavage site after BAL 31 exonuclease digestion. This probe was end labeled by using Klenow enzyme and [a-32P]dCTP. To facilitate construction of the Ap.mb and 3'SP8 inserts, the XbaI site found immediately upstream of the .m poly(A) site was converted into a Sall site. As a consequence, a second probe was required to accommodate the XbaI-to-Sall site conversion. This probe is a 290-bp SalI-HpaI fragment that contains 173 bp of p.-5 intergenic sequence plus 111 bp of vector sequence. The probe was end labeled with Klenow enzyme and [a-32P]TTP. The b-specific probe is a 350-bp AccI-PvuI fragment containing 174 bp of C83-encoding sequence with an additional 47 bp of down-

5342

TISCH ET AL.

stream intron sequence plus 119 bp of vector sequence. The 8 probe was end labeled with Klenow enzyme and [a-32P]dATP. The SV40 early-gene poly(A) site-specific probe is a 245-bp XbaI-BamHI fragment isolated from the pECE vector and end labeled with Klenow and [a-32P]dCTP. The neo-specific probe is a 1,400-bp NcoI-HpaI fragment end labeled with Klenow enzyme and [a-32P]dCTP. Hybridization temperatures for the various probes were as follows: 173-bp ILm poly(A), 36°C; 290-bp Um poly(A), 43°C; 8, 47°C; SV40 poly(A), 34°C; and neo, 52°C. Si nuclease digestion was for 30 min at 40°C. The reactions were analyzed on either 3.5 or 8% polyacrylamide-8M urea gels. Transcription analysis of isolated nuclei. Transcriptional analysis was carried out essentially as described by McKnight and Palmiter (19), with some modifications. In brief, approximately 3 x 107 isolated nuclei were incubated with 200 ,uCi of [ox-32P]UTP for 15 min at 30°C for each assay. Nuclei were lysed in a solution containing 0.5 M NaCl, 0.05 M MgCl2, 0.002 M CaCl2, and 0.01 M Tris (pH 7.4) and incubated with DNase I (10 ,ug/ml) at 37°C for 20 min. The reaction was deproteinized by digestion with proteinase K (100 jig/ml) at 37°C for 30 min, phenol-chloroform extracted, and applied to a Sephadex G-50 column. Pooled nuclear RNA fractions were partially hydrolyzed by incubation in 0.1 N NaOH on ice for 10 min. Excess amounts of M13 single-stranded DNA probes (5 ,ug) were immobilized on GeneScreen Plus with a dot blot apparatus. 32P-labeled RNA (5 x 106 cpm) was hybridized with the filter for 48 h at 65°C. The blots were washed with 0.1 x SSC (SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 65°C and treated with RNase A (10 ,ug/ml). Relative hybridization to each probe was evaluated by densiometric readings and normalized for probe size. Inserts encompassing the ji-8 transcriptional unit were subcloned into either M13mpl8 or M13mpl9 DNA. The M13 probes used were as follows: probe 1, a 910-bp XbaI-HindIII VHTNP fragment; probe 2, a 351-bp PstI-EcoRI fragment containing VH-C,U1 intron sequence; probe 3, a 1,210-bp HindIII fragment containing C,u3-C,u4 sequence; probe 4, a 737-bp KpnI-PstI fragment containing Ml exon sequence; probe 5, a 171-bp PstI-XbaI fragment containing M2 exon sequence; probe 6, a 520-bp HindIII-PstI fragment; probe 7, a 570-bp PstI-EcoRI fragment, and probe 8, a 440-bp EcoRIBgIIII fragment spanning the inm-8 intron region; probe 9, a 1,231-bp BglII-SstI fragment containing CbM- and CH-encoding sequences; probe 10, a 970-bp BglII-BamHI fragment containing BM2 sequence; probe TK, a 855-bp BamHIHindIlI noncoding fragment upstream of a CK light-chain gene; and probe pBR, a 379-bp EcoRV-SphI pBR322 fragment. All probes lack repetitive sequences. RESULTS Analysis of tim poly(A) site utilization in M12 transfectants. A model system was devised in which the sequences required for preferential use of a ji poly(A) site relative to 8-gene expression could be assessed. For this purpose, the vector pRC8s was constructed with a unique Sall restriction site between a VH gene segment encoding for TNP-hapten binding (VHTNP) and a B-chain gene. Sequence variants of the jim-8 intron region containing a PUm poly(A) site could then be introduced between VHTNP and 8-gene sequences (Fig. 2). The C,u-encoding region was not included to facilitate the construction of the various plasmids. The design of the jLm-8 insertion constructs permitted direct assessment of the role of splice signals associated with the Pm8 intron in

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modulating cleavage at the IXm poly(A) site and splicing between VHTNP and Cb1 exons. The p.n-8 insertion plasmids were stably transfected into a variant of the B lymphoma M12, which represents an early stage of B-cell development. This M12 variant, unlike the parental line, does not express endogenous immunoglobulin at a transcriptional level (12). The RNA expressed from these plasmids was analyzed by both Northern (RNA) blotting and S1 nuclease mapping. In transfectants expressing the Lmb plasmid, which carries the complete PLm-8 insert (Fig. 2), a single 1.2-kb VHTNP-specific transcript was detected (Fig. 3). The size of the transcript indicated that a VHTNP-tOILm exon splice had occurred and that the ,m poly(A) site was utilized. To map the cleavage site of this transcript, an end-labeled probe containing the ,Lm poly(A) site (Fig. 4) was hybridized to M12 Lmb cytoplasmic mRNA and digested with S1 nuclease. As expected, a 116-nucleotide protected fragment that was detected in mRNA from the PUm heavy-chain-expressing pre-B cell line 70Z/3 (Fig. 4) was also observed in the M12,umb transfectant mRNA, con-

IMMUNOGLOBULIN 8 HEAVY-CHAIN GENE EXPRESSION

VOL. 10, 1990

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firming that the authentic Pm poly(A) site was cleaved. The lack of detectable 8 mRNA in the U transfectants appeared to be the direct result of posttranscriptional processing, since b-gene transcription was observed by in vitro transcriptional analysis (Fig. 5 and 6). These data suggest that sequences necessary for efficient p.m poly(A) site utilization are present within the imb8 intron insert. Various studies have demonstrated that the efficiency of

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TISCH ET AL.

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poly(A) site usage can be modulated by sequences 3' of the site (2, 8). Two repetitive elements and a 162-bp uniquesequence inverted repeat (USIR) are found downstream of the poly(A) site (27). These sequences may establish an RNA secondary structure that facilitates poly(A) site usage. To investigate whether these 3' sequences are involved, all but 30 bp of sequence found downstream of the cleavage site was deleted in the [Lm30+8 plasmid. This 3' deletion did not impair poly(A) site usage (Fig. 4), and the 1.2-kb VHTNp-P,m-specific transcript (Fig. 3) was expressed. Splicing signals are required for ULm and SV40 poly(A) site usage. To examine the possibility that splicing influences the efficiency of the cleavage event, three mutants, M25, A[Lmb, and 3'SP8 (Fig. 2), were generated in which the normal splicing process has been altered. In M25, the Ml exon and associated splice acceptor have been deleted. This deletion alters the normal sequence of splicing events in which the M2 splice acceptor and not the Mi-associated site interacts with the incoming donor site. In AP.mj, both 1Lm-encoding exons and all associated splice signals have been removed, poly(A) site and downstream sequences leaving the P.m

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intact. The P.m exons have been replaced with a 3' splice acceptor site in the 3'SP8 construct. Analysis of M28 transfectants demonstrated that removal of the Ml exon did not impair Plm poly(A) site usage. A single mature VHTNPspecific transcript 1.0 kb in length was detected, indicating that a VHTNp-to-M2 exon splice had occurred (Fig. 3). Furthermore, a 116-nucleotide Si nuclease digestion product was observed (Fig. 4), suggesting that appropriate P.m poly(A) site cleavage had taken place. No 8 mRNA could be detected. Interestingly, when all splicing signals associated with the P.m poly(A) site were removed from the AuLPmB plasmid, cleavage at the P.m poly(A) site did not occur (Fig. 4) and 5-encoding mRNA could be detected (Fig. 3 and 4). A 3' terminus could not be cleaved nuclear transcript with a detected, ruling out the possibility that such a message was synthesized but inefficiently transported from the nucleus to the cytoplasm in the AP.mj transfectants (Fig. 4). These results indicate that splicing may have a critical role in poly(A) site selection. This notion was confirmed upon analyzing the 3'SPS transfectants, in which the addition of a 3' splice acceptor site upstream of the PLm poly(A) site facilitated cleavage (Fig. 4). This cleavage event resulted in Pm

P.m

VOL. 10, 1990

5345

IMMUNOGLOBULIN 8 HEAVY-CHAIN GENE EXPRESSION

the expression of a 0.9-kb VHTNp-encoding transcript, thereby preventing 8 mRNA synthesis (Fig. 3). This result suggests that a 3' splice acceptor site located between the VHTNP- and R,uw-encoding exons is required for 1m poly(A) site utilization. To determine whether the requirement for splicing signals is unique to the Um poly(A) site, we generated plasmid IXmSV408, in which an SV40 early-gene poly(A) site was inserted between the VHTNP and 8 genes. As seen with the AULmb transfectants, the SV40 poly(A) site was not utilized, resulting in VHTNp-specific 8 mRNA expression. However, when the SV40 poly(A) site was ligated downstream to either the P'm exons (tLmSV408) or the CK 3' splice acceptor site (3'SPSV408), cleavage occurred (Fig. 3 and 4). In 3'SPSV408 transfectants, the SV40 poly(A) site was used inefficiently, resulting in some 8 mRNA synthesis. The expression of 8 mRNA may reflect an inefficient VHTNp-to-3' CK acceptor splice event, leading to reduced usage of the SV40 poly(A) site. Alternatively, the inefficient use of the SV40 poly(A) site in the 3'SPSV408 transfectants may be due to the weakness of the SV40 poly(A) site relative to the lm poly(A) site. Interestingly, in 3'SPSV408 transfectants of the terminally differentiated B cell line J558L, the SV40 poly(A) site was efficiently utilized and no 8 mRNA was detected (data not shown). These results suggest that the generation of transcripts containing P1m or SV40 3' termini requires a splice signal upstream of the respective cleavagepolyadenylation sites. The Lm.-8 intron region can regulate transcription termination. It has been demonstrated that in IgM-secreting B cells, termination by RNA polymerase occurs in the vicinity of the Um exons (7, 9, 18, 32). The signal(s) governing this transcription termination is ill defined. To ascertain whether the Um8b intron region can direct the termination process seen upstream of the 8 gene in IgM-secreting B cells, in vitro transcription assays were carried out on nuclei isolated from J558L plasmacytoma cells transfected with the various ±m-8 insertion constructs. The J558L plasmacytoma (23) was used as a model of a terminally differentiated cell. These cells no longer express endogenous immunoglobulin heavy-chain mRNA and therefore allow analysis of expression from the insertion constructs without background immunoglobulin gene transcription. Drug-selected J558L clones expressing VHTNP-specific mRNA determined by RNA dot blotting were pooled so as to eliminate potential clone-specific artifacts. Mature 8 mRNA, as determined by Northern blot analysis, was detected only in the AIJm8 and pRCbs transfectants (data not shown). Transcription termination was assayed in the transfectants by using 10 different singlestranded probes extending throughout the entire Um-b transcription unit (Fig. 5). The transcriptional profiles of the transfectants shown in Fig. 6 represent hybridization of these probes after normalization relative to the VH probe. As models of appropriate transcription termination within the PLm_8 unit in terminally differentiated cells, IgM-secreting M2 myeloma cells and J558 pRpu-8 transfectants that expressed only R,u mRNA as determined by Northern blot analysis were analyzed. As expected, transcription of the 8 gene was less than 10% of that detected with VH-region probes. In both cell lines, the majority of termination by RNA polymerase, approximately 70 to 80%, occurred within a 1,243-bp region found just downstream of probe 5 and spanning probe 6, which contained the p.m poly(A) site. Transcription diminished further over the following 560 bp (probe 7). A similar transcriptional profile was seen in the Rm8 transfectants, suggesting that the absence of upstream C,u sequences did

TABLE 1. Summary of transcription level detected in the 8 gene (probe 9) upon removal or replacement of sequences found in the p.-8 intron region 8 gene (probe 9) Sequence

transcriptiona

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0.06 0.15

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0.64

,mSV408 ..........................

0.59 0.32

................................

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0.65

0.69 0.59

not alter the termination event. In control M12 L,mb transfectants and in the B lymphoma WEHI-231, which represent immature stages of B-cell maturation, transcription termination did not occur within the 1,243-bp region described above. Instead, significant levels of transcription extended further downstream to a 444-bp segment (probe 8) found immediately 5' of the 8 locus, suggesting that termination in the J558L ALmb transfectants is subject to developmental control. Interestingly, termination by RNA polymerase within the 444-bp segment was evident even in transfectants expressing plasmid pRCbs, which carries no additional WUm~8 intron sequences. The reduced rate of transcription within the 8 gene may explain why in the pRCbs transfectants the amount of 8 transcript, as judged by the relative signal with the VH probe (Fig. 3), was less than that of ,um-terminated transcripts. Marked transcription termination within the 444-bp segment has previously been noted in splenic B cells and in B cells derived from long-term bone marrow cultures (33), implicating a termination site not subject to developmental control. Sequences upstream of the termination region are required for efficient transcription termination. To identify whether other regions are involved in the termination process, an additional set of plasmids was constructed in which sequences upstream and downstream of the 1,243-bp terminating region found between probes 5 and 6 were either deleted or replaced. In the P,umTK8558 plasmid, 855 bp of downstream sequences containing the USIR were replaced with noncoding sequences found 5' of the CK light-chain gene. The USIR has the potential of forming a stem-loop (8), thereby influencing DNA secondary structure. Transcriptional analysis of the p.mTK8558 transfectants indicated that transcription termination still occurred within the 1,243-bp region found downstream of probe 5 and spanning probe 6, despite the replacement of the USIR and downstream sequences (Fig. 5 and 6). Removal of the majority of the ,u-8 intron and in turn the 1,243-bp termination region by either deleting sequences downstream of the p.m poly(A) site (except for 30 bp) in the ,um30+8 plasmid or replacing the PLm poly(A) site and downstream sequences with the SV40 poly(A) site in the FLmSV408 plasmid resulted in levels of transcription in probe 9 that were 64 and 59%, respectively, of the level of transcription detected by the VH probe (Table 1). These results coupled with those obtained with the 11mTK8558 transfectants suggest that the 1,243-bp region found between probes 5 and 6 is essential for appropriate termination of RNA polymerase. When either the ILm exons were completely removed (AWLmb) or a 700-bp segment found 200 bp upstream of the termination region was replaced (pBRILmb), only 20 to 30% RNA polymerase downloading

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was observed within the 1,243-bp region (Fig. 5 and 6). Furthermore, when the p.m exons were replaced with the CK 3' splice acceptor signal in the 3'SP8 plasmid, the level of transcription in probe 9 was 69% of that detected by the VH probe (Table 1). These results suggest that a splicing signal in the absence of upstream sequences is not sufficient to mediate appropriate transcription termination. Therefore, it appears that sequences downstream of the 1,243-bp terminating region encompassed by probes 5 and 6 have only a minor role in the transcription termination process, whereas 5' sequences are essential for the event.

DISCUSSION Using a set of plasmids in which U intergenic sequences were inserted upstream of a 8 gene and transfected into the B lymphoma M12, we have shown that 8 mRNA expression from these constructs is regulated by a splicingdominant mechanism. Furthermore, the complete pm.b intron insert used in this study is sufficient to regulate transcription termination within a 1,243-bp region upstream of the 8 gene in transfectants representing a terminally differentiated stage of B-cell maturation. By ablating the splicing capacity of the poly(A) site containing inserts, we were able to show that the Rm cleavage event is compromised, resulting in 8 mRNA synthesis in M12 transfectants. Restoration of the cleavage event is seen when an appropriate splice acceptor signal is found upstream of the pm poly(A) site. Furthermore, an SV40 early-gene poly(A) site is functional only when p.-encoding exons and associated splice signals or a CK gene splice acceptor signal are inserted upstream. These results argue against a mechanism whereby poly(A) site selection determines the appropriate splicing pattern. Splicing may facilitate cleavage and polyadenylation by making the poly(A) site accessible to the appropriate factors. Alternatively, since both p.m and Cbl splice acceptor sites compete for the same VHTNP gene splice donor site, a VHTNP-to-,m splice would eliminate the VHTNP-tO-Cbl splice which otherwise effectively competes with the p.m poly(A) site cleavage reaction. The mechanism that regulates the VH-to-C81 splice event in transcripts encoded by the insertion plasmids may not be entirely the same as that utilized for transcripts of the naturally occurring p.-8 gene. The VH and Cbl splice signals on the natural p.-8 transcripts are separated by a 13-kb intron that contains the p. gene and a number of alternative splice sites. The B-cell stage-specific joining of the VH and Cbl splice signals is thought to be promoted in part by transacting factors that selectively bring the two sites together. On the other hand, the shortened intron (approximately 5 kb) and the lack of alternative splice signals in the plasmid constructs might allow the VHTNP-to-Cbl event to proceed and effectively compete with the R.. cleavage-polyadenylation reaction in the absence of such trans-acting factors. A number of examples have recently been published that demonstrate that splice site selection or a commitment to a specific splice site before the actual event governs poly(A) site usage. These examples include the tissue-specific expression of calcitonin versus CGRP mRNA (14) and the developmental expression of E3 versus L4 mRNA in the adenovirus (1). Work by a number of groups (4, 24, 30, 31) and most recently by Peterson and Perry (25) has shown that regulated splice site choice is an important component of the ps-versus-p.m poly(A) site selection process. Collectively, the data suggest that competition exists between polyadeny-

MOL. CELL. BIOL.

lation at the proximal R,u poly(A) site and a splice between C,u4 and Ml exons. The two events are mutually exclusive in that cleavage at the p, poly(A) site prevents a Cp.4-to-Ml splice, whereas the splice event effectively removes R,p terminus-encoding sequences and the associated poly(A) site. The results obtained in this study support the notion that a splice event is linked to p,, poly(A) site usage. The data presented above suggest that a hierarchy may exist in which active selection of splice sites influences choices between p. or 8 mRNA synthesis and between R,, or p,, poly(A) site selection. The determining event in the selection of p. or 8 mRNA synthesis may involve initial V.-to-Cp.l or VH-to-C8l splice events. For example, in early B cells a committed splice complex or actual VH-tO-Cp.l splice would effectively preclude 8 mRNA synthesis and possibly promote additional ,u-specific mRNA processing events. In support of this notion, Nelson et al. (21) have noted that splicing of VH to the C,ul exon frequently precedes additional 3' splicing events in p.-specific precursors. In mature B cells, a VH-to-Cbl splice would be promoted, possibly by preventing the VH-tO-Cp.l splice event. A trans-acting factor(s), for example, could bind to the Cp.1 exon splice acceptor signal, sequester additional p. splice sites, and in turn make the VH splice donor signal accessible to CM1 splice signals. If a VH-to-Cbl splice were to take place on a growing transcript, this could explain why a full length p.-8 primary transcript has yet to be identified (18). Work is currently in progress to ascertain whether such a mechanism can indeed regulate 5-gene expression. Analysis of the J558L pRp.-8 transfectants and the M2 myeloma cells demonstrated that transcription termination occurs predominantly downstream of the p.m exons within a 1,243-bp region which contains the p.m poly(A) site. These results confirm recent findings of Law et al. (13), who also demonstrated that termination takes place within this region in M2 cells. Furthermore, an analogous transcription profile was seen for the J558L Rmb transfectants, suggesting that this construct provides a valid model system with which to study the termination process in terminally differentiated B cells. Our findings contradict those obtained in a recent study in which J558L cells transfected with a vector carrying a complete p. gene, minus the 8 gene, exhibited marked transcription termination prior to the p.m exons (9). The discrepancy in termination site localization may reflect differences in the constructs used in the two studies. The absence of a 8 gene downstream of the p.-encoding locus in the other construct may effect high-order structure involving, for example, a chromatin conformation that favors a termination event 5' of the p.m exons. Expression studies of eucaryotic genes transcribed by RNA polymerase II have suggested that transcription termination can be influenced by a number of sequence elements that may, for example, be involved in RNA and DNA secondary structure or binding of trans-acting factors (26). Our study demonstrates that the 1,243-bp region alone is insufficient to direct the termination process. When upstream sequences are either removed (ApLmb) or replaced (pBRp,mS), the efficiency of the termination event within the 1,243-bp region is significantly reduced. Recent studies evaluating transcription termination in the mouse P-globin (17) and adenovirus ElA (6) genes have revealed that cleavage of the nascent transcript is necessary for efficient RNA polymerase downloading. The high level of transcription detected through the p.m-8 region in the Ap.mS transfectants may in part be due to impaired pm poly(A) site cleavage. Interestingly, in the pBRRumb and 3'SPS transfec-

VOL. 10, 1990

IMMUNOGLOBULIN 8 HEAVY-CHAIN GENE EXPRESSION

tants in which the p.m poly(A) site is cleaved (data not shown), transcription termination is still disrupted, suggesting that cleavage-polyadenylation alone is not sufficient for appropriate termination of RNA polymerase. What function the sequences located upstream of the 1,243-bp region may have is uncertain. These sequences may serve by destabilizing the elongating RNA polymerase complex. Alternatively, the DNA secondary structure of the termination region may be disrupted by the replacement sequences sufficiently to limit the accessibility of putative DNA-binding trans-acting factors required for termination. Recent work by Law et al. (13) has revealed a nuclear factor-binding site 30 bp upstream the IUm poly(A) site. The binding of a factor in this region could directly impede the progress of the RNA polymerase molecule and aid transcription termination. Consistent with this idea is the recent finding of Connelly and Manley (3) that a CCAAT promoter element, known to be a site for DNA-binding proteins, is necessary for termination in an adenovirus-encoding vector. Whether the nuclear factor identified by Law et al. (13) can still bind to its DNA recognition sequence in pBRp.m8 transfectants is now being determined. The study of transcription termination within the Ptm-B intergenic region provides an excellent model system for assessing regulatory aspects of this process. Using the constructs generated in this study, we hope to identify sequences within the 1,243-bp region that have a functional role in transcription termination in addition to surmising the importance of RNA cleavage for this process. This type of analysis will ideally lead to the identification and isolation of termination-specific factors. ACKNOWLEDGMENTS We thank H. P. Nguyen for expert technical assistance. We are indebted to A. Roach and J. Kang for critical comments. R.T. holds a Medical Research Council studentship. This work was supported by the National Cancer Institute. LITERATURE CITED 1. Adami, G., and J. R. Nevins. 1988. Splice site selection dominates over poly(A) site choice in RNA production from complex adenovirus transcription units. EMBO J. 7:2107-2116. 2. Berget, S. M. 1984. Are U4 small nuclear ribonucleoproteins involved in polyadenylation? Nature (London) 309:179-182. 3. Connelly, S., and J. L. Manley. 1989. A CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal. Cell 57:561-571. 4. Danner, D., and P. Leder. 1985. Role of an RNA cleavage/ poly(A) addition site in the production of membrane-bound and secreted IgM mRNA. Proc. Natl. Acad. Sci. USA 82:86588662. 5. Ellis, L., E. Clauser, D. 0. Morgan, M. Edery, R. A. Roth, and W. J. Rutter. 1986. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 45:721-732. 6. Falck-Pederson, E., J. Logan, T. Shenk, and J. R. Darneli. 1985.

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and processing of the messenger RNAs specifying heavy and light chain immunoglobulins in MOPC-11 cells. Cell 15:14951509. 29. Thomas, P. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5205. 30. Tsurushita, N., N. M. Avdalovic, and L. J. Korn. 1987. Regulation of differential processing of mouse immunoglobulin p. heavy-chain mRNA. Nucleic Acids Res. 15:4603-4615.

MOL. CELL. BIOL. 31. Tsurushita, N., and L. J. Korn. 1987. Effects of intron length on differential processing of mouse p. heavy-chain mRNA. Mol. Cell. Biol. 7:2602-2605. 32. Yuan, D., and P. W. Tucker. 1984. Transcriptional regulation of the p.-8 heavy chain locus in normal murine B lymphocytes. J. Exp. Med. 160:564-583. 33. Yuan, D., and P. L. Witte. 1988. Transcriptional regulation of p. and 8 gene expression in bone marrow pre-B and B lymphocytes. J. Immunol. 140:2808-2814.

Parameters that govern the regulation of immunoglobulin delta heavy-chain gene expression.

The mu and delta immunoglobulin heavy-chain genes comprise a complex transcriptional unit in which a single mRNA precursor gives rise to mu- and delta...
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