Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
1 Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
Correspondence to: D. Yuan
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Abstract |
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Keywords: B lymphocytes, transcription termination, polyadenylation, IgD, IgM, internal ribosome entry site
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Introduction |
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In this study, we have utilized two mutant mouse strains to investigate the regulation of transcription termination within the µ gene complex. The pµ.µ
Ratt strain harbors a transgene in which a 1160 bp µ
intergenic segment, termed att, is inverted. This segment contains the putative cis-regulatory region necessary for transcription termination upstream of the
exons (20,21) in immature B cells. In the µS/ strain the µS exon, along with its polyadenylation site, has been deleted in the genomic DNA resulting in a complete deficit of secretory IgM production (22). The deletion does not include the region containing a conotical binding site for MAZ, a zinc finger protein originally isolated by virtue of its binding to a site within the c-myc P2 promoter. Occupancy of this site has been postulated to cause DNA bending resulting in polymerase pausing (23) and therefore may participate in the regulation of termination within the µ
gene complex. The use of primary B cells provides the opportunity for examination of the possible additional effect of changes in extent of polymerase initiation during different stages of B cell differentiation. This difference (24,25), not detectable in tumor cells (26,27), may alter the ability of polymerases to recognize pause/termination sites. By transcription analysis of the µ
gene complex in these two strains we show that whereas both polyadenylation as well as cis-regulatory sequences are important for transcription termination, the mode of action of the sequences depends on the stage of B cell differentiation.
Because run-on transcription analyses of transfectants are cumbersome and sometimes results are difficult to interpret, we designed a novel method to assess transcription read-through in an attempt to further limit the att site sequences. Using this method, we have identified a 200 bp segment from the att site that induces transcription termination as effectively as the entire full-length segment in early B lineage cells but not in mature B cells. Correspondingly we have also demonstrated B cell stage-specific proteins that bind to this gene segment.
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Methods |
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Animals
A SalIIBsaAI fragment (see Fig. 2A) isolated from the pµ.µ
Ratt construct, devoid of plasmid sequences, was microinjected into mouse oocytes of the CD-1 strain (Jackson Laboratories, Bar Harbor, ME) by the Microinjection Services of UTSW Medical Center. Offspring were typed for transgene insertion by Southern blot analysis of tail DNA utilizing a
M specific probe. Because of the insertion of cDNA sequences in the construct, genomic hybridizing fragments could be distinguished from fragments containing the transgene. Two of these animals, lines 4393 and 4399, successfully transmitted the gene to the offspring. B cells in these two lines displayed similar phenotypes. The founders were mated with C57BL/6 (Jackson Laboratories). Further typing of transgene in offspring of founder mice was performed by PCR amplification utilizing primers containing sequences from
MI (5'-GTGACAGCTACATG) and
MII (5'-AGACCACAGCATGCTT) which amplified a 380 bp fragment from the transgene and a 600 bp fragment from the endogenous gene. The transgenic mouse line carrying a rearranged Ig heavy chain gene with µ and
encoding regions in the genomic context (MD-3, 29) was obtained from Dr Christopher Goodnow (John Curtin School of Medical Research, Canberra, Australia) and bred to C57BL/6 mice in our facilities. Mice carrying the transgene were typed by FACS staining with
a and µa reagents as described below. The generation of the µS/ mouse line (22) has been previously described.
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In vitro `run-on' analysis
Nuclei were prepared from cells and nascent RNA was labeled for 15 min at 30°C as previously described (2) using 100 µCi of [-32P]UTP (3000 Ci/mol; ICN Radiochemicals, Irvine, CA) per sample. Following the labeling, RNA was extracted and hybridized at 42°C for 3 days to 5 µg of double-stranded DNA probes immobilized on nitrocellulose. Subsequently, the filters were washed, treated with RNase as previously described, and exposed to a PhosphorImager screen and quantified using the Imagequant software package (Molecular Dynamics, Sunnyvale, CA). After subtraction of background hybridization to vector alone, relative hybridization to each probe was divided by the size of the probe to assess relative polymerase loading on each gene segment.
Probes used for run-on analysis
Plasmid DNA containing gene fragments as previously described (25) and listed below were used for run-on analysis of bone marrow cells. This is possible because the DNA fragments introduced into mouse eggs contained no plasmid sequences, as evidenced by low hybridization to the control KS plasmid with no insert. (#1) The VD probe (21) is a 400 bp PCR product generated from the Pµ. vector and cloned into pBluescript II (KS, Stratagene, La Jolla, CA). (#2) The 5' Cµ probe is a 550 bp TagIPstI fragment from cDNA of MOPC 104 µ mRNA (34) containing part of CH2, all of CH3 and part of CH4 subcloned into pGEM4 (Promega, Madison, WI). (#3)The 3' Cµ probe is the adjoining 440 bp cDNA fragment containing part of CH4, the µS exon up to the end of the µS processed mRNA subcloned into pGEM4. (#4) The µSµM intronic probes is a 346 bp KpnIHaeIII genomic fragment subcloned into pGem2. (#5) The µMI probe is the adjoining 385 bp HaeIIIPst1 genomic fragment subcloned into pGem 2. (#6) The µMII probe is the adjoining 330 bp PstIIHincII fragment genomic containing the µMII exon and all of the 3' untranslated region of µM mRNA subcloned into pGEM4. Because the µS/ transgene contains a deletion of the intronic region between µS and µM and between the two µM exons, the value of the probe size used for normalization was reduced accordingly. (#7) The 5' att probe is a 345 bp XhoIPst1 genomic fragment, located in the µ intron, subcloned into pGEM4. (#8) The 3' att probe is the adjoining 550 bp PstIEcoRI genomic fragment subcloned into pGem 4. (#9) The C1-3 probe is a 764 bp fragment subcloned from TEPC 1017 cDNA into pUC 8 containing C1,
H and C3. (#10). The
M probe is a 710 bp HincIIBglII genomic fragment containing
M1 and
MII, subcloned into pGEM-2. Because the pµ.µ
Ratt transgene has a deletion of the MIMII intron, the value of the probe size used for normalization was reduced accordingly. (#11) The GAPDH probe is a 1233 bp complete rat cDNA (35) cloned into KS. The C
probe is a 482 bp HpaIBglII genomic fragment including the C
exon subcloned into pUC 8.
FACS
Peripheral blood lymphocytes or bone marrow cells were analyzed on the FACScan flow cytometer (Becton Dickinson, San Jose, CA) using reagents previously described (21).
Plasmid construction for luciferase assay
pGLIRES
This vector was created by modification of the pGL3-basic (Promega, Madison, WI). The SalI site at 2010 was first destroyed before insertion of the internal ribosomal entry sites (IRES) from EMCV virus into the BglIINcoI site. The IRES sequence (36,37) was amplified from pCIN4 (Dr Stephen Rees, Glaxo Wellcome) with the primers 5'-CGAGCATAGATCTAGGGCGGCGAATTCG-3', incorporating BglII and EcoRI sites (underlined) and 5'-GGGGTTGTGCCATGGTTATCATCGTGTT-3' containing NcoI sites (underlined).
pGLIgµ
A 1084 bp DNA segment containing 460 bp 5' of µM I and extending 140 bp 5' of the µMII poly(A) signal was amplified from a vector containing the genomic sequences using the primers 5'-ACGCACGGGAATTCTGATCAAGAAAGT-3', containing EcoRI site (underlined) and 5'-CATCAGAATTCACTGGTCGACTTCCAGT-3' incorporating EcoRI and SalI sites (underlined). The PCR product was digested with EcoRI and cloned into EcoRI site of PGLIRES to generate PGL3µM. The rearranged VDJ segment and IgH promoter region was then amplified from pµPoly (27) with the primers 5'-GGCGTATCAGAGGCCCT-3' which anneals to upstream of IgH promoter and 5'-GGTCAGTCTAGAAAGAAGACCATC-3' which anneals within µMII. The PCR product was digested with SalI and BclI and blunt-ended by Klenow fill-in and cloned into SmaI site of pGL3µM to generate pGLIgµ.
pIgµfIVS or pIgµrIVS
A1200 bp gene segment containing the µ intronic sequences spanning the PstI site 3' of µMII up to 25 bp 5' of the C1 acceptor splice site was excised from the pµMSUSIR+ vector (20) and ligated into the SalI site of pGLIgµ in both orientations. Vectors containing subfragments of the `IVS' region (pGL5'IVS, pGL3'IVS, pGL3'IVSx and pGL3'IVSy) were all derived by subcloning the fragments as shown in Fig. 4
(B) into the SalI site of pGLIgµ.
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Luciferase and ß-galactosidase assays
Luciferase activity was determined by using the Luciferase Assay Kit from Promega (Madison, WI) according to the manufacturer's instructions and measured with Optocomp luminometer (MGM Instrument). For the ß-galactosidase assay, 100 µl of cell extract was diluted in 50 µl of 1xCell Lysis Buffer (Promega) and 150 µl of 2xAssay Buffer (0.2 M sodium phosphate buffer, pH 7.3, 2 mM MgCl2, 0.1 M ß-mercaptoethanol, 1.33 mg/ml ONPG) was then added. Reactions were incubated at 37°C until the absorbance could be read at 420 nm. Luciferase activity was normalized to ß-galactosidase activity in each transfection to correct for transfection efficiency.
Nuclear extract preparation
Nuclear extracts from TD1.1 or J558L cells were prepared by slight modifications of previously described methods (39).
Gel electrophoresis mobility shift assays
DNA probes were either generated by restriction enzyme digestions of subclones of the `IVS' region as indicated in Fig. 4(B) or by PCR amplification from the plasmid template containing the relevant sequences. Oligonucleotides used to generate the PCR fragment for the EcoRIAccI region were: forward primer 5'-CAGGGAAAGAACAGAATTCTG and reverse primer: 5'-ATAAGTGTAGACCTGTGACAC. These primers were in turn used to generate the F105 and F88 fragment by using two additional oligonucleotides located in the middle of the segment. These are: forward primer 5'-AATTGGATGATGAACCCTGA and reverse primer: 5'-CCAGAGTCACCTCTATGT. Products of the appropriate size were isolated by agarose gel electrophoresis and 5'-end-labeled with [
-32P]ATP (4500 Ci/mmol) and T4 polynucleotide kinase. Binding reactions contained 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 20% glycerol, 100 mM KCl, indicated amount of poly(dI)poly(dC) heteropolymer (Pharmacia), 5 mM DTT and 10,000100,000 c.p.m. of end-labeled probe. Reactions initiated by the addition of protein were incubated for 30 min at 23°C. ProteinDNA complexes were resolved by PAGE containing 90 mM Trisborate (pH 8.3), 2 mM EDTA and 2% glycerol. The running buffer was the same as the gel buffer except that glycerol was omitted. Gels were dried and exposed to phosphorimaging screens.
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Results |
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The µS polyadenylation site induces transcription termination
We had previously observed that in activated B cells transcription termination occurs at a region significantly more upstream of that occurring in early B cells. The transcription profile obtained from day 4 LPS-activated splenic B cells from 4399 mice (Fig. 2C) shows that this alteration of the site of transcription termination is not compromised by the inversion of the att site. Termination of the majority of the transcripts in activated B cells occurs within the µMII exon. There also appears to be pausing of polymerases in regions immediately preceding this termination site. This profile does not differ from that found for LPS-activated non-transgenic B cells (Fig. 3C
). We had previously observed that the site of termination in activated B cells is variable depending on the state of activation (25). Because of the preferential usage of the µS poly(A) site in activated B cells, however, it is not clear whether the termination is a consequence of this usage or whether activation of termination-inducing trans-regulatory factors, such as, for example, the MAZ protein, results in the use of the upstream poly(A) site by default. To distinguish between these possibilities, we sought to determine whether termination in the µM region occurs in B cells obtained from the µS/ mouse strain which cannot utilize the µS poly(A) site because it has been deleted. It should be noted that the µM poly(A) site and immediate downstream regions were not disturbed, therefore the binding of possible termination factors to the template or nascent RNA that extends through this segment should not be affected. The transcription profile of non-stimulated splenocytes from these µS/ mutant animals was similar to that of normal littermates (Fig. 3B
and data not shown). Upon LPS activation the relative level of hybridization between the µMII probe (#6) versus the Cµ probe (#2) did not change. In contrast, in LPS-activated cells from littermate controls there was a significant decrease in this ratio. Thus, the termination usually found within this region was much alleviated. Interestingly, the pronounced pausing of polymerases in the region defined by the µMI probe often found in non-transgenic B cells also occurred in the µS/ strain despite the deletion of the µMIµMII intron. Significantly, although polymerases fail to exit in the region defined by probe #6, the majority of the newly induced transcripts in the mutant still terminates in a more upstream region than that in resting B cells.
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Luciferase assay to measure transcription termination efficiency
Despite the alleviation of transcription termination in bone marrow cells of animals harboring the Pµ.µRatt transgene to a level that is approximately the same as in mature spleen cells (Fig. 2C
) the extent of processing of the transcripts to the C exons is not increased. Further analysis indicated that splicing of VDJ to the C1 exon is diminished even in mature B cells (manuscript in preparation). Correspondingly, no IgD+ cells can be detected in the bone marrow (Fig. 1B
) and a significant fraction of peripheral B cells express only IgM in the absence of IgD (see Fig. 1A
). The alteration in processing cannot be attributed to deletion of part of the
gene in the 3' end (Fig. 2A
) because this phenotype was not observed in the strain harboring the pµ.µ
att transgene which has an identical 3' configuration (21). The disruption of downstream processing appears to be due to the mutation of the att insert, therefore it is necessary to further refine the limits of this cis-regulatory element so that a change therein can be targeted exclusively to transcription termination.
IRES have been employed to produce polycistronic mRNAs in retroviral-mediated gene transfer systems (37). Here we use IRES from the encephalomyocarditis virus genome, a member of the picornaviridae family of viruses (36,37), for efficient expression independent of the 5' cap site of transcripts. The strategy is to construct a series of transfection vectors that contain putative termination sites located upstream of an IRES element such that only read-through transcripts that extend into the luciferase gene can be translated by ribosomes that enter at the internal ribosome binding sites and produce luciferase protein. In the absence of the luciferase promoter transcripts that terminate before the IRES will not extend to the coding sequence for the luciferase gene and no enzyme can be produced. In order to first test if the presence of IRES itself affects luciferase expression, we compared luciferase activity derived from the pGL3p control and the pGL3pIRES vector which has an IRES sequence inserted between the SV40 promoter and luciferase coding region. Figure 4(A) shows that in the presence of IRES (pGL3pIRES) luciferase expression in a plasmacytoma cell line (J558L) was decreased by 4560% of that generated by direct transcription/translation from the SV40 promoter (pGL3p control). This level of decrease is not dissimilar to the effect of IRES shown previously in other systems (42,43) and the signal intensity is still sufficient for use as a reporter system. We then substituted, for the SV40 promoter in pGL3pIRES, a gene segment that includes the Ig heavy chain promoter together with rearranged VDJ sequences as well as the µM coding region with intact poly(A) signal (pGLIgµ; Fig. 4B
).
To ascertain that the level of transcription read-through as measured by this method is similar to that measured by run-on transcription analysis we first tested the effect of inserting the entire 1200 bp `IVS' sequence previously shown to induce transcription termination (20). The pIgµfIVS vector was transfected in parallel with control plasmid pGLIgµ and the relative luciferase activity derived from each plasmid was compared. Two cell lines representing two extreme ends of the B cell differentiation spectrum were transfected. The TD1.1 lymphoma cell line is derived from a pre-B cell culture and J558L is a plasmacytoma. Figure 4(C) shows that in TD1.1 cells luciferase activity derived from pIgµfIVS was <40% of that derived from the control plasmid, indicating that more than half of the polymerases that transited the µM region failed to reach the IRES and luciferase gene. However, in J558L cells the same fragment showed little effect on the abilities of polymerases to read through IRES and the luciferase gene. Moreover, transfection of pIgµrIVS, in which the 1200 bp `IVS' segment was inserted in the reverse orientation, into either TD1.1 or J558L cells demonstrated comparable luciferase activity to that derived from the control plasmid in both cell lines showing that the termination efficiency of this segment is orientation dependent as described previously. These results are comparable to those from previous in vitro run-on analyses and confirm both the validity of the assay and the functionality of the terminator present in the µ
intronic region in the context of the IRESluciferase construct.
Deletion analysis of the µ intergenic region
In order to further delineate the elements required for regulation of termination, various fragments in the `IVS' region were generated by restriction enzyme digestion and inserted into pGLIgµ and the effect of each fragment was assessed for termination efficiency. Figure 4(D) shows that luciferase expression from pIgµ3'IVS which contains the 3' half of `IVS' region was decreased to 50% of that from pGLIgµ, while luciferase expression from pIgµ5'IVS which contains the 5' half of `IVS' region did not differ from the control vector. These results suggest that the EcoRIBglII fragment within the `IVS' region plays an important role in transcription termination. This fragment was further bisected and cloned separately. Analysis of these two constructs, pGLIgµ3'X and pGLIgµ3'Y, showed that the 5' half of active EcoRIBglII fragment decreased luciferase activity as effectively as the entire fragment, whereas the 3' half did not significantly affect termination. Thus, these results demonstrate that essential elements for termination reside within the 200 bp fragment cloned into pGLIgµ3'IVS.
Protein binding to the fragment required for termination
To understand the mechanism by which termination may be dictated by the 182 bp EcoRIAccI fragment within the pGLIgµ3'IVS we assessed whether proteins could bind to it in a B cell stage-specific manner. This fragment was 5'-end-labeled with [-32P]ATP and binding reactions were performed by incubating the probe with either TD1.1 or J558L nuclear extracts followed by analysis of DNAprotein complexes on a non-denaturing polyacrylamide gel. Complexes with two different mobilities were formed by nuclear extracts from TD1.1 cells but not by those from J558L cells (Fig. 5A
), whereas a control fragment derived from a gene segment located more upstream but still within the `IVS' region yielded complexes of similar mobility when bound to nuclear extracts from either TD1.1 or J558L cells (Fig. 5B
). The complex C1 with slower mobility was derived from specific binding because of its susceptibility to competition by the specific competitor consisting of the unlabeled probe but not by a non-specific competitor (Fig. 5C
), whereas the complex with the faster mobility, C2, which appeared with greater variability, was competed away by both specific and non-specific competitors. Other representative cell lines at similar developmental stages were also screened for the binding activity. As shown in Fig. 5
(D), Bine 4.8, another pre-B cell line, demonstrated a similar binding activity as that found for TD1.1 cells, whereas a lower level of binding was found in extracts from M12.4, a lymphoma line representing an intermediate stage of B cell differentiation.
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In an effort to further localize the protein binding site we bisected the EcoRIAccI fragment by PCR amplification using two sets of primers resulting in adjacent 5' 105 bp and 3' 88 bp fragments (F88 and F105). When each of these fragments was separately assessed, F88 fragment formed complexes resulting in four bands with extracts from TD1.1 cells. Only the band with the slowest mobility was concentration dependent and showed reproducible binding (Fig. 6A) and could be effectively competed by the homologous unlabeled fragment (Fig. 6B
) but to a much lesser extent by F105. The faster mobility bands were competed by both homologous and heterologous fragments. In contrast, labeled F105 exhibited much lower levels of binding activity for the same extract. Whether the strong binding to the 88 bp fragment can account for the total termination activity of the pGL3'IVSx vector still awaits further functional confirmation.
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Discussion |
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This observation is consistent with recent findings showing that an increase in abundance of the 64 kDa subunit of the cleavage stimulation factor, CstF64, in activated B cells may be an important (50) but not sole driving force (51) for the selection of the µS poly(A) site. Interestingly, the site of termination upon increased µS polyadenylation is located >2 kb downstream of the cleavage site. Therefore, despite the dependence on polyadenylation, this process cannot be classified as poly(A) directed (19). In contrast, in repeated run-on transcription assays (25; see also Fig. 2D), we have noted large variations in the extent of polymerase loading, which can often be much higher than that in the Cµ region, in the probes encompassing the region upstream of the µM poly(A) site. Therefore it appears that there is a polymerase pause site just downstream of this region that causes variations in the extent of polymerase entrapment. The best candidate for this site is the conotical MAZ binding site (23) located 200 bp downstream of the µM poly(A) site within a region previously documented to be a polymerase unloading site in tumor cells (4,52). The termination of transcription close to a pause site located some distance from the polyadenylation site is consistent with a model whereby polymerase activates polyadenylation (46) by continued association with these processing factors (45). The presence of the MAZ site may provide the specificity needed for targeting of the increased amount of functional CstF64 to the IgH gene.
Regardless of the possible involvement of the MAZ site in activated B cells, clearly this site is bypassed in mature, resting B cells so that the exons are transcribed. In contrast, analysis of the pµ.µ
Ratt strain shows that in the presence of the att site, which is located further downstream, in the appropriate orientation, is necessary for transcription termination in immature B cells. Much of the polymerase downloading is effectively prevented by the insertion of this intergenic segment in the wrong orientation. The data presented herein does not address the question of whether the µM poly(A) site is necessary for termination at this site in primary B cells although previous data in transfectants indicated a necessity for the site. However, recent studies in a new gene knockout mutant offer some insight into this question. The
polypeptide chain has been shown to have the ability to completely replace µ chain function during early B cell development in the IgM/ mouse (53) which harbors a deletion of the entire µ gene as well as the att sequence located upstream of the
exons. Interestingly, in the µMT/ mouse strain (54) in which only the µMI exon was replaced by a neo gene, the presence of the downstream
gene does not replace µ gene function in early B cells. It appears, therefore, that the retention of the att sequence in this case effectively prevented transcription progression to the
exons despite the presence of the strong poly(A) site used by the inserted neo gene transcript. IgD expression from the mutated chromosome can be found in mature B cells of heterozygous animals, showing that the effect of the termination site can be overcome upon B cell maturation. Hence even in the presence of a heterologous polyadenylation signal the att sequence can function as an attenuation site.
Using a novel assay for assessing transcription termination we have defined the minimal cis-regulatory sequences within the att region that mediate termination. This assay is much more sensitive than run-on transcription analysis because it does not rely upon measurement of hybridization signals derived from labeled nascent transcripts. By this means we have defined a 200 bp segment within the att region that can induce transcription termination which, in the genomic context, is located 1650 bp downstream of the µMII poly(A) site and 650 bp 5' of C1. Of special significance is the apparent differentiation stage specificity of the ability of this segment to dictate transcription termination. Thus, termination is registered only in tumor cells that represent the equivalent of immature B cells, whereas in the plasmacytoma line, J558L, representing more mature B cells, the segment does not affect polymerase transit. Because termination activity is based on comparison with relative read-through in the parental vector that also contains the µM poly(A) site, any termination directed by this site should not affect the read-out. For all of the vectors analyzed, the level of termination detected by this method is, however, still incomplete for reasons that are unclear. It is possible that there are further upstream as well as downstream elements not included in the construct that exert additional modulatory effects. Nevertheless, these results establish the sequences contained within the 200 bp EcoRIAccI fragment as a minimum requirement for induction of termination.
The mechanism underlying the termination-inducing activity of the EcoRIAccI fragment remains unclear. One clue is derived from the correlation of protein binding with the functional activity of the fragment. We have shown that nuclear extracts from transformed counterparts of early B cells can bind to this fragment, whereas much less activity is contained with extracts from more mature B cell equivalents. Furthermore, specific binding can be restricted further to a 88 bp sequence. A simplistic model would predict that the protein binding serves as a block to transcription read-through and this binding is altered upon B cell activation. Further characterization, including footprint analysis, will be necessary in order to show whether there is preferential binding to the coding strand. However, the orientation dependence of the effect on transcription suggests that the att sequence does not function by bending DNA as has been shown for MAZ (23). Furthermore, from the transcription profile there is no evidence for the region to act as a strong pause site for polymerase. Finally, it is always important to bear in mind that in vitro assays performed in transformed B cells do not always correlate completely with in vivo mechanisms occuring in normal B cells.
In conclusion, the findings reported here allow us to present a comprehensive view of transcription across the µ gene complex. In early B cells, the µM poly(A) site is preferentially utilized to produce cells expressing exclusively membrane IgM. Usage of this site does not induce significant transcription termination despite the presence of the MAZ site located immediately downstream. It is not until the att site located some 1650 bp downstream is encountered that polymerase downloading occurs. We had previously designated this region as an attenuation site (21) because, in contrast to a termination site, the recognition of the att site is reversibly regulated by B cell maturation such that to allow read-through to downstream C exons, the site must be bypassed. We have now further refined the limits of this att site. At the other end of the differentiation spectrum occurring upon B cell activation, transcription initiation of the IgH gene complex is increased. The corresponding increased loading of polymerases may augment the effectiveness of the MAZ site resulting in the accumulation of polymerases in the immediate upstream region. The enhanced cleavage of the newly induced transcripts at the µS poly(A) site due to the increase in the polyadenylation factor CstF64 must destablize the polymerases to an extent that results in the dissociation of the majority of the transcripts when they reach a relatively discrete site within the µM 3'-untranslated region.
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Acknowledgments |
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Abbreviations |
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IRES | internal ribosome entry site |
LPS | lipopolysaccharide |
µS | secretory form of IgM |
µM | membrane form of IgM |
PE | phycoerythrin |
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Notes |
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Received 4 November 1998, accepted 2 February 1999.
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References |
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