Structure of Germline Immunoglobulin Heavy-Chain γ1 Transcripts in Interleukin 4 Treated Mouse Spleen Cells

Antibody class switching is mediated by a DNA recombination event that replaces the Cμ gene with one of the other heavy (H) chain constant region (CH) genes located 3' to the Cμ gene. The regulation of this process is essential to the immune response because different CH regions provide different biological functions. Correlative evidence indicates that the isotype (class) specificity of the switch is determined by the accessibility of specific CH genes as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germline CH genes is unknown. In this report, we describe the structure of RNA transcribed from unrearranged γ1 genes in mouse spleen cells treated with LPS plus .a HeLa cell supernatant containing recombinant interleukin 4. The germline γl RNA is initiated at multiple start sites 5' to the tandem repeats of the γ1 switch (Sγ1) region. As is true for analogous RNAs transcribed from unrearranged γ2b and c genes, the germline γ1 RNA has an exon transcribed from the region 5' to Sγ1 sequences, which is spliced at a unique site to the Cr gene. The germline γ1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acid in length.


INTRODUCTION
A functional immunoglobulin (Ig) heavy (H) chain gene is comprised of several gene segments that are brought together by gene rearrangement during differentiation of antibody-producing cells. The antigen binding site is encoded by variable (V), diversity (D), and joining (J) segments that are initially .associated with the C constant region gene. After immunization, the same VDJ gene may subsequently be expressed with other downstream H-chain constant (CH) region genes by a process called class switching, thus changing the effector function of the antibody while maintaining its antigen specificity. Class switching occurs by a DNA recombination mediated by repetitive sequences known as the switch (S) regions that lie a few kilobases upstream of each CH gene (except C). A VDJ gene initially *Corresponding author. associated with the C, gene can be translocatecl to a downstream CH gene by switch recombination occurring between the S, region and the S region of a target CH gene (reviewed in Grimacher, in press).
Aspects of the process of class switching can be studied in cultured cells. Normal IgM / splenic B cells from mice will switch to IgG and IgGab expression after polyclonal stimulation in culture with lipopolysaccharide (LPS) (Bergstedt-Lindqvist et al., 1984). T-cell-derived lymphokines can influence the isotype to which the cells switch (Isakson et al., 1982;Bergstedt-Lindqvist et al., 1984Coffman et al., 1988;Lebman and Coffman, 1988). The addition of interleukin 4 (IL-4) to lipopolysaccharide (LPS) induced spleen B cells stimulates switching to IgGl and IgE and suppresses switching to IgG and IgG2b. Several lines of evidence indicate that IL-4 directs the switch recombination to the yl and e genes. For example, IL-4 has been shown to increase the frequency of precursors for IgG1 + and IgE / cells 12 M. XU AND J. STAVNEZER (Bergstedt-Lindqvist et al., 1988;Coffman et al., 1988;Lebman and Coffman, 1988). IL-4 appears to direct the switch to yl and e by increasing the accessibility of the yl and e genes, as shown by the fact that IL-4 induces RNAs transcribed from unrearranged yl and e genes and reduces the level of RNA transcribed from unrearranged 72b genes prior to the expression of IgG1 and IgE Rothman et al., 1988;Stavnezer et al., 1988;Berton et al., 1989;Esser and Radbruch, 1989). Although the function of transcription of the germline CH genes is unknown, strong correlative evidence indicates that only unrearranged CH genes that are transcriptionally active are capable of undergoing switch recombination (Stavnezer-Nordgren and Sirlin, 1986;Yancopoulos et al., 1986;Rothman et al., 1988;Stavnezer et al., 1988;Berton et al., 1989;Esser and Radbruch, 1989;Severinson et al., in press). RNA transcription may simply be a by-product of the accessibility of CH genes to switch recombinase. Alternatively, transcription might serve as part of the mechanism of switch recombination or the transcripts or a polypeptide encoded by them might direct class switching. To identify DNA sequences necessary for their regulation and to understand the possible function(s) of germline transcripts, it is first necessary to know the structure of the germline transcripts. We report here the structure of yl germline transcripts induced in mouse spleen cells by treatment with LPS plus a HeLa cell supernatant containing rIL-4. We find that the structure of the yl germline RNA is similar to that of RNAs transcribed from unrearranged Cy2b and Ca genes Radcliffe et al., 1990).

LPS and IL-4 Induce Germline yl RNA Transcripts
We have previously shown that treatment of spleen cells with IL-4 or a supernatant from a TH2 cell line that contains IL-4 (Noma et al. 1986) (and other factors in the presence or absence of LPS induces in B cells transcripts from unrearranged Crl genes that hybridize with the 5'Sr HindIII-PstI segment ( Fig.   1A) as 1.7-kb and 3.2-kb RNA species (Stavnezer et al., 1988;Severinson et al., in press). We began to localize the sequences encoding the exon of germline yl RNA by additional RNA blotting experiments. The 2.7-kb HindIII-PstI fragment derived from clone py1/EH10 (Mowatt and Dunnick, 1986) was subcloned into three fragments: a 1.2-kb HindlII-BamHI fragment, a 0.8-kb BamHI-KpnI fragment, and a 0.7-kb KpnI-PstI fragment (Fig.lA).
Labeled RNA probes transcribed from these fragments were hybridized with blots containing poly(A) + RNA from mouse spleen cells induced for 2 days with LPS and IL-4. Of these three fragments, only the KpnI-PstI probe detected the 1.7-kb and 3.2-kb RNAs (Fig. 1B). No RNA was detected by the HindIII-BamHI or BamHI-KpnI probes (data not shown). No RNA was detected with a KpnI-PstI probe for antisense transcripts (data not shown). This result indicates that the exon of germline yl RNA is encoded within the KpnI-PstI fragment.
Determination of Splice Site of Germline yl RNA In order to precisely locate the Ir exon, we used the PCR to prepare cDNA clones containing the 3' donor splice site of the I exon. Based on the RNA blotting data described before and previous work (Stavnezer et al., 1988), we expected that the germ- Using the PCR to amplify cDNA products from poly(A) + RNA from spleen cells treated for 2 days with LPS and 15% rIL-4-containing HeLa cell supernatant, we obtained several cDNA clones that should contain the splice site of germline yl RNA. Eight of these clones were sequenced. All eight of these clones had the identical splice donor located between nucleotides 633 and 634 in Fig. 2A and demonstrated that the splice acceptor at the 5' end of the Cr gene, which is used in yl mRNA, is used for germline yl RNA. The location of the Ir splice donor is consistent with RNase protection experiments in which a predominant protected band of 281 bp was obtained after hybridization of total cell RNA with a RNA probe transcribed from the BglII-PstI segment (data not shown). These results are also consistent with $1 protection experiments of Berton et al. (1989). The   RNase-resistant fragments obtained after hybridization of the RNA probe used in B electrophoresed alongside a DNA-sequencing ladder. Lanes are P, probe alone; Y, probe hybridized with yeast RNA (10g); and S, probe hybridized with total cell RNA (10 g) from spleen (treated as in B). (D) Products of primer-extension experiment using oligo 2 electrophoresed alongside a DNA-sequencing ladder. Lanes are O, oligonucleotide incubated alone; Y, primer extension with yeast RNA (150/g); and S, primer extension with total cell RNA (150 g) from spleen (treated as in B).

Initiation Sites of Germline yl RNA
The initiation sites for germline 71 RNA were determined by RNase-protection and primer-extension experiments. In a RNase-protection experiment, hybridization of RNA from spleen cells (induced with LPS plus 15% HeLa cell supernatant) with a RNA probe transcribed from the genomic DNA KpnI-PstI segment produced multiple bands after electrophoresis of the RNase resistant products on a DNA-sequencing gel (Fig. 1C). The lengths of the predominant bands varied from 387 to 484 nucleo-tides. Less predominant bands of139 to 359 nucleotides in length were also observed. These results indicated that the 5' border of the I, exon occurred at multiple sites since we had only found a single 3' splice site, suggesting that the germline 71 RNA may have heterogeneous initiation sites. To confirm this and to more precisely locate the initiation sites, primer-extension experiments were performed. As the RNase-protection experiments indicated the major initiation sites are located 5' of the BglII site, an oligonucleotide (oligo 2) complementary to the sequence from 8-24 nucleotides 3' to the BglII site  Fig. 2A) was used for the primer-extension experiments. The sizes of the primer-extended products (Fig. 1D) matched with those predicted from RNaseprotection experiments and indicated that there are multiple initiation sites for germline yl RNA. The fact that the results from RNase protection and primer extension consistently and completely corresponded indicated that the multiple bands on the gels were not due to the degradation of the RNA. In addition to the predominant initiation sites located 5' to the BglII site, there are several initiation sites located 3' to the BglII site that were detected by the RNase protection assay shown in Fig. 1C and by primer-extension experiments (not shown) using an oligonucleotide (oligo 1) complementary to sequences located 75-92 nucleotides 5' of the splice site. Taken together, the germline yl RNA has multiple initiation sites distributed over a 345nucleotide region, but the predominant initiation sites are located within a region of about 97 bp at the 5' end of the I1 exon. The most 5' initiation site of the I1 exon is 484bp upstream of. the Irl/Cr splice site. The sizes of the It1 exon determined from these experiments (387 to 484 bp) (predominant) and the Cr exon (1067bp) (Honjo et al., 1979) would produce a germline yl RNA of 1.7 kb, assuming a 200-nucleotide poly(A) tail. This corresponds in size to the predominant 1.7-kb germline yl RNA detected on RNA blots. As is also true for analogous RNAs transcribed from immunoglobulin c (Radcliffe et al., 1990), y2b , and / (Lennon and Perry, 1985) genes, there are no TATA or CCAAT boxes located within 150 nucleotides upstream of the initiation sites of the germline yl RNAs ( Fig. 2A).
The Germline yl RNA Has an Open-Reading Frame (ORF) The nucleotide sequence of the Ir exon indicated the presence of an open-reading frame (ORF) that would be initiated by a Met codon located in such a position that germline yl RNA initiated at any of the sites we detected would have this Met. Potentially, this ORF would encode a 48 amino acid polypeptide with a termination codon located within the Crl domain ( Figs. 2A and 2C). The reading frame that would be ,used in the C rl exon for the ORF in germline RNA differs from that used in the mRNA for yl H chains. The predicted amino acid sequence of the ORF is indicated below the nucleotide sequence in Fig. 2C. As true for the ORF encoded by germline c RNA, the nucleotide sequence surrounding the initiator AUG codon should form a good translation initiation site according to Kozak (1986,1987) since it has a purine at the -3 and a G at the +4 positions.

DISCUSSION
The germline RNA transcribed from unrearranged C H genes may simply be an indicator of the accessibility of CH genes. Alternatively, the act of transcription, the RNAs themselves, or their products may function in class switching. The facts that each of the unrearranged CH genes (except C) have been shown to be transcribed under appropriate conditions (Stavnezer-Nordgren and Sirlin, 1986;Stavnezer et al., 1988;Severinson et all, in press), and that where examined, these transcripts have an exon located 5' to the S region, and that transciption proceeds through the S region in the sense direction Stavnezer et al., 1988;Berton et al., 1989;Radcliffe et al., 1990) suggests that this transcription has a function in class switching. Additional common properties are that the germline y2b , c (Radcliffe et al., 1990), / (Lennon and Perry, 1985) and 1 RNAs all have multiple initiation sites and no TATA or CCAAT boxes located 5' of their initiation sites. Some of the initiation sites of y2b, /z, and yl RNA have similar nucleotide sequences (Fig. 2D), suggesting that they may use a common transcription factor. However, these sequences differ from the initiator sequences that have been defined for other genes that lack CCAAT and TATA boxes (Sehgal et al., 1988;Smale and Baltimore, 1989).
The germline 1 and cz RNAs differ from the y2b and / germline RNAs in that the 1 and a transcripts have small ORFs with Met initiation codons in contexts that should allow relatively efficient translation, whereas the y2b and / germline RNAs have small ORFs with Met codons in poor contexts for translation according to Kozak (1986Kozak ( , 1987. The germline c RNA, which has an ORF encoding a 43 amino acid protein (that differs from the sequence encoded by the yl ORF), appears to be located on small polysomes in 1.29/ B lymphoma cells and this association is enhanced by LPS treatment (.Radcliffe et al., 1990). LPS treatment induces class switching from IgM / to IgA / in 1.29/ cells (Stavnezer et al., 1985). Thus, it is interesting to speculate that c and yl germline RNAs encode polypeptides that function in class switching, but this is probably not the only function of these transcripts, since it appears likely that not all germline RNAs are translated.
The determination of the initiation sites and structure of germline yl RNA allows us to begin studies to define the DNA regions necessary for regulated expression of the RNAs. It will be important to define these sequences in order to understand how heavy-chain switching is regulated.

MATERIALS AND METHODS
Mice and Cell Culture BALB/c mice were purchased from Charles River Breeding Laboratories (Wilmington, Massachusetts). Spleen cells (2 xl06/ml) from mice were cultured for 2 days in RPMI 1640 (GIBCO, Grand Island, New York) in the presence of 10% fetal calf serum (HyClone Laboratories, Logan, Utah). LPS (RIBI Immunochem Research Inc., Hamilton, Montana) was added at 25/,g/ml. Either 15% HeLa cell supernatant (Bergstedt-Lindqvist et al., 1984), which contains IL-4 and other interleukins, or 8 units/ml of recombinant IL-4 were also added (Noma et al., 1986) (kindly donated by Eva Severinson of the University of Stockholm).

RNA Isolation and Blot Hybridization
Total cell RNA was prepared by the guanidinium isothiocyanate-CsC1 protocol and poly(A)+ RNA was isolated by one cycle of chromatography on oligo(dT)-cellulose. Radioactive RNA probes were transcribed from 5'Srl germline DNA fragments cloned into Bluescript plasmids (Stratagene, La Jolla, CalifOrnia) and hybridization was performed as described (Maniatis et al., 1982).
The amplified products were cloned into a Bluescript plasmicl with blunt-end ligation and identifield using the 5'Sr BamHI-PstI fragment as a probe (Fig. 1A).

RNase Protection
Full-length 32p-labeled antisense RNA probes were transcribed by T7 polymerase from Bluescript plasmids containing various 5'S1 segments. RNase protection analysis was performed using these probes as described (Zinn et al., 1983), except that nuclease P1 (20/g/ml) was used instead of RNase A.