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The Journal of Immunology, 1999, 163: 4375-4382.
Copyright © 1999 by The American Association of Immunologists

Transcriptional Regulation of the Ig{kappa} Gene by Promoter-Proximal Pausing of RNA Polymerase II1

Eva E. Raschke, Thomas Albert2 and Dirk Eick3

Institute for Clinical Molecular Biology and Tumor Genetics, GSF National Research Center for Environment and Health, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transcriptional regulation can occur at the level of initiation and RNA elongation. We report that the rearranged, nontranscribed Ig{kappa} gene in the pre-B cell line 70Z/3 harbors a paused RNA polymerase II (pol II) at a position between 45 and 89 bp downstream of the transcription initiation site. LPS, an inducer of NF-{kappa}B, activated Ig{kappa} gene transcription by increasing the processivity of pol II. TGF-ß inhibited the LPS-induced transcription of the Ig{kappa} gene, but not initiation and pausing of pol II. A rearranged copy of the Ig{kappa} gene was introduced into 70Z/3 cells using an episomal vector system. The episomal Ig{kappa} was regulated by LPS and TGF-ß like the endogenous gene and established a paused pol II, whereas a construct with a deletion of the intron enhancer and the C region did not establish a paused pol II. Two distinct functions can therefore be assigned to the deleted DNA elements: loading of pol II to its pause site and induction of processive transcription upon LPS stimulation. It had been proposed that somatic hypermutation of Ig genes is connected to transcription. The pause site of pol II described in this work resides upstream of the previously defined 5' boundary of mutator activity at Ig{kappa} genes. The possible role of pausing of pol II for somatic hypermutation is discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The murine B cell lymphoma line 70Z/3 serves as a model for Ig{kappa} gene activation during the differentiation of pre-B cells into B cells (1, 2). 70Z/3 cells constitutively produce Ig heavy chain mRNA and harbor a functionally rearranged Ig{kappa} gene that is not transcribed. LPS induces expression of the Ig{kappa} gene in 70Z/3 cells (1, 3). The transactivation of the Ig{kappa} gene by LPS requires the activation of NF-{kappa}B and the binding to the Ig{kappa} gene intron enhancer ({kappa}Ei) (4, 5) that is located in the intron between the J cluster and the C region (6, 7). In addition to the binding site for NF-{kappa}B, which is crucial for {kappa}Ei function, the {kappa}Ei contains an octamer motif, a binding site for {kappa}BFA, E boxes, and, adjacent, a matrix attachment region (MAR)4 (8, 9). A second enhancer in the Ig{kappa} gene has been identified downstream of the C region ({kappa}E3') (10). The {kappa}Ei is required and sufficient for Ig{kappa} gene activation in pre-B cells, whereas the {kappa}E3' enhancer, either alone or in synergy with {kappa}Ei, is important for Ig{kappa} gene expression during later differentiation stages (11, 12, 13, 14).

Additionally to the transcriptional activation of Ig genes (15), Ig gene enhancers initiate and coordinate other processes at the Ig gene loci. This includes induction of chromatin accessibility (16), demethylation of the chromosomal locus (17), rearrangement of V, D, and J regions (18, 19), as well as the later somatic hypermutation of the VJ region (20).

Heat-shock genes and the protooncogenes c-myc and c-fos belong to a growing group of genes that are regulated by promoter-proximal pausing of pol II (21, 22, 23, 24). At these genes, a pol II initiates and transcribes a short stretch of RNA, but then pauses at a site proximal to the promoter. Gene induction mediated by transcriptional activators confers processivity to the paused pol II (25). Pol II density at the pause site remains unchanged during gene induction, indicating that induction of processive transcription is tightly coupled with initiation and loading of pol II onto the pause site.

In Burkitt lymphoma and mouse plasmacytoma, Ig gene loci are frequently translocated to the c-myc gene, resulting in its constitutive activation (26). Because Ig enhancers activate the translocated c-myc by conferring processivity to the paused pol II (27, 28), we asked whether Ig enhancers activate Ig gene transcription by a similar mechanism. In this study, we show that pol II pauses proximally to the transcription start site of a functionally rearranged Ig{kappa} gene. Reconstitution of this promoter-proximal pausing of pol II on stably transfected episomal constructs revealed that a fragment containing the {kappa}Ei/MAR and C region is necessary for the establishment of a paused pol II complex at the Ig{kappa} gene promoter. LPS treatment stimulates a late step in Ig{kappa} gene transcription and mediates induction of processive transcription of paused pol II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

70Z/3 cells were maintained at 37°C and 5% CO2 in RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Life Technologies). For better growth, 1 mM sodium pyruvate (Life Technologies), 50 µM {alpha}-thioglycerol, and 20 nM bathocuproinedisulfonic acid (Sigma, St. Louis, MO) were added to the medium as reducing agents. Cells were treated with 15 µg/ml LPS from Salmonella typhosa (Sigma), 2 ng/ml TGF-ß (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT), or 20 µg/ml cyclosporin A (Sigma). For establishment of polyclonal cell lines, 5 x 106 cells were electroporated with 20 µg of DNA at 250 V and 960 µF (Bio-Rad, Richmond, CA) and subsequently split for 4 wk under selection with 300 µg/ml hygromycin (Calbiochem, La Jolla, CA). For each construct, two polyclonal cell lines were established with two independently derived plasmids. Raji and mouse erythroleukemia cells were cultivated and transfected like 70Z/3 cells, with the exception that they were grown in the absence of reducing agents.

Primer extension

Primer extension analysis was performed with 20 µg of total cellular RNA of 70Z/3 cells grown for 24 h in the presence or absence of LPS. The primer used had the sequence 5'-GAA GCA CTG ATT AGC AGG AAG CTG-3' corresponding to nucleotides 799–822 of the VJ region antisense strand (29). Labeling of the primer, hybridization, and reverse transcription was done as described (30).

Preparation of nuclei

After washing cells twice with ice-cold PBS, the pellets of 108 cells were resuspended in 25 ml lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). After incubation on ice for 5 min, the lysate was spun down at 1500 rpm for 15 min at 4°C. The pelleted nuclei were resuspended in storage buffer (50 mM Tris-HCL, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA) and immediately frozen in liquid nitrogen in portions of 100 µl, corresponding to 2 x 107 nuclei.

In vivo footprinting

Nuclei were exposed to freshly prepared 10 mM KMnO4 for 2 min at 37°C. Reactions were stopped by addition of 1 ml lysis buffer (300 mM LiCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 2% w/v SDS, 200 µg/ml proteinase K, 2% 2-ME) and incubated at 55°C for 2 h. In vitro modification of DNA, purification, and cleavage at modified base residues were done essentially as described previously (31). All DNAs were subjected to linker/ligation-mediated PCR (32). Primers used to footprint the Ig{kappa} promoter region had the following sequences: V{kappa}1, 5'-TAT CTT GCG ATT TGC ATA TTA CAT TTT CAG-3' (nucleotides 660–689); V{kappa}2, 5'-CAT ATT ACA TTT TCA GTA ACC ACA AAT ATC TC-3' (nucleotides 674–705); and V{kappa}3, 5'-AAC CAC AAA TAT CTC ACA GTT GGT TTA AAG C-3' (nucleotides 691–721) corresponding to the Ig{kappa} gene sense strand (29). Hybridization temperatures were 50°C for V{kappa}1 (18 PCR cycles), 60°C for V{kappa}2 (18 cycles), and 66°C for V{kappa}3 (3 cycles). Examination of T residues in the antisense strand failed for technical reasons, because the sequence between nucleotides +120 and +270 of the Ig{kappa} gene comprising intron sequences is extremely AT rich (>75%), not allowing the selection of appropriate primer combinations for ligation-mediated PCR.

Nuclear run-on assay

Nuclei were thawed on ice, mixed with 100 µl run-on buffer (10 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 300 mM KCl; 0.5 mM each of ATP, GTP, and UTP; 100 µCi [{alpha}-32P]CTP 800 Ci/mmol), and incubated for 15 min at 28°C. A total of 5 µl DNase I (Boehringer Mannheim, Indianapolis, IN; RNase free) was added, and the incubation was continued at room temperature for 10 min. After addition of 15 µl 10 mg/ml proteinase K and 5 µl 10% SDS, the samples were incubated at 37°C for 2 h. Nuclear transcripts were separated from unincorporated nucleotides on a Sephadex G-50 column equilibrated in TE. The labeled run-on RNA was hybridized to oligonucleotides immobilized on a nylon membrane at 60°C for 2 days in hybridization buffer (400 mM Na2HPO4, 100 mM NaH2PO4, 7% SDS, 1 mM EDTA, pH 8). The oligonucleotides used for the respective run-on analysis are described (Fig. 2GoC and Fig. 5GoA). Filters were subsequently washed in buffer 1 (1% SDS; 0.1x SSC), followed by incubation in buffer 2 (1 mM EDTA; 2x SSC; 4 µg/ml RNase A) for 30 min at room temperature. After a second wash in buffer 1, the filters were dried and exposed to x-ray films.



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  		FIGURE 2. Unpaired T residues proximal to the transcription start site of the Ig{kappa} gene. A, Mapping of the transcription start site. Total cellular RNA of untreated 70Z/3 cells (lane 3) or cells treated with LPS for 24 h (lane 4) was subjected to primer extension analysis. A sequence ladder of A residues (lane 1) and T residues (lane 2) of the template strand served as size reference. The transcription start site and the location of the primer are indicated. B, In vivo footprinting of KMnO4-sensitive T residues. Genomic DNA (lane 2) or isolated nuclei of 70Z/3 cells (lanes 3 and 4, cultivated without and with LPS for 24 h, respectively) were treated with KMnO4. After cleavage at the oxidized residues with piperidin, ligation-mediated PCR was used to amplify the DNA fragments, followed by gel electrophoresis. A ladder of G residues served as reference (lane 1). The positions of the hypersensitive T residues are indicated at the right-hand side. C, Sequence of the promoter-proximal region of the Ig{kappa} gene in 70Z/3 cells. The octamer site, the TATA box, and the translation start site are marked with boxes. The transcription start site is marked by an arrow; oligonucleotide A starts with +1. Arrows indicate the location of hypersensitive T residues, as derived from the footprint experiment. The sequences corresponding to the run-on oligonucleotides Up and A–F are indicated.

 


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  		FIGURE 5. Pol II distribution within promoter-proximal sequences of the episomal Ig{kappa} gene. A, Sequence of the promoter-proximal region of the episomal Ig{kappa} gene. The octamer site, the TATA box, and the translation start site are marked with boxes. The transcription start site is marked by an arrow. The location was estimated according to the observation that the octamer site in Ig{kappa} genes usually lies ~70 bp upstream from the transcription initiation site (29 ). The sequences corresponding to the run-on oligonucleotides Up' and A'–D' are indicated. B, Nuclear run-on experiment with established cell lines. Nuclei were isolated from cell lines containing pBPV-Ig{kappa} (lane 1) and pBPV-Ig{kappa}{Delta} (lane 2). Run-on reactions were performed and labeled RNAs were hybridized to a set of five antisense oligonucleotides Up' and A'–D' comprising the region immediately upstream and downstream of the transcription start site of the episomal Ig{kappa} gene. The 7SK oligonucleotide served as a pol III transcription control. C, Control for hybridization efficiency. Uniformly labeled RNA of the episomal Ig{kappa} gene transcribed in vitro by T7 RNA polymerase was hybridized to the run-on oligonucleotides Up' and A'–D'.

 
Preparation of in vitro transcribed RNA by T7 RNA polymerase

For production of a uniformly labeled RNA specific for the 70Z/3 endogenous and the episomal Ig{kappa} gene, DNA fragments encompassing the respective Ig{kappa} gene sequences were fused to the T7 RNA polymerase promoter by PCR. In vitro transcription by T7 RNA polymerase was done in presence of [{alpha}-32P]CTP (800 Ci/mmol) essentially as recommended by the manufacturer. Full-length transcripts were isolated by preparative PAGE and used for hybridization to oligonucleotides, as described above.

Northern blot analysis

RNA was prepared from 70Z/3 and Raji cells using RNeasy (Qiagen, Chatsworth, CA). Northern blot analysis was performed as described (30). A total of 20 µg total RNA was loaded per lane. The probes were labeled with [{alpha}-32P]dCTP. For probe Ig{kappa}, a HindIII/BamHI cut fragment containing the {kappa}Ei/MAR and C region of the mouse Ig{kappa} gene from clone T1 was used (33). A VJ region-specific probe for the 70Z/3 endogenous Ig{kappa} gene was isolated from genomic DNA by PCR amplification (Boehringer Mannheim) from nucleotides 609-1332 in regard to the published sequence of the 70Z/3 Ig{kappa} gene (29). The primers used for the sense and antisense strands were 5'-GGG CAC ATG AAA TAC TGA GAA TGG TG-3' and 5'-ATT TCC AGC TTG GTG CCT CCA CCG-3', respectively. A hybridization probe for the episomal Ig{kappa} gene was prepared by purification of the EcoRI/XbaI fragment from clone T1 containing the region from bp -838 to +806.

Construction of the vectors

pBPV was generated in a two-step process. Linker 1, introducing a XhoI, SalI, SfiI, and BamHI site, was ligated to the ClaI/SalI fragment of p.Rep4 (Invitrogen, San Diego, CA), thereby destroying the SalI site. This was followed by insertion of the full-length BPV genome (pBPV; Pharmacia, Piscataway, NJ) into the BamHI site, resulting in plasmid pBPV. For pBPV-Ig{kappa}, the EcoRI/BamHI fragment from clone T1 containing the Ig{kappa} gene (33) was cloned into the SalI site of linker 1 using blunt end ligation with Klenow enzyme. pBPV-Ig{kappa}{Delta} was constructed by insertion of the EcoRI/HindIII fragment of clone T1 into the SalI site in pBPV. pEBV was obtained by cleavage of pRF261-4 (27) with HindIII/BamHI, followed by religation of the 10.6-kb fragment containing EBNA1/oriP for episomal replication, a hygromycin resistance gene, and sequences for amplification and selection in bacteria. For construction of pEBV-Ig{kappa}{Delta}, the 10.6-kb BamHI/HindIII fragment of pRF261-4 was ligated to the XhoI/HindIII fragment of pBPV-Ig{kappa} by combining the BamHI and the XhoI site by blunt end ligation, thus introducing 840 bp of the Ig{kappa} promoter and sequences upstream of the {kappa}Ei/MAR into pEBV (see Fig. 4GoA for location of restriction enzyme sites). pEBV-Ig{kappa}{Delta} was cut with XbaI/HindIII, and the XbaI/SfiI fragment of pBPV-Ig{kappa} containing the {kappa}Ei/MAR and C region was inserted, resulting in pEBV-Ig{kappa}.



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  		FIGURE 4. Transcriptional regulation of the endogenous and the episomal Ig{kappa} genes. A, The constructs used for transfection. pBPV contains the full-length BPV sequence, the hygromycin resistance gene as a selectable marker, and sequences for amplification in Escherichia coli. The Ig{kappa} and Ig{kappa}{Delta} genes shown below were inserted into pBPV, resulting in pBPV-Ig{kappa} and pBPV-Ig{kappa}{Delta}. B, Total cellular RNA was prepared from 70Z/3 cells (lanes 1, 2, 11, and 12) and cell lines containing pBPV (lanes 3, 4, 13, and 14), pBPV-Ig{kappa} (lanes 5–8 and 15–18), and pBPV-Ig{kappa}{Delta} (lanes 9, 10, 19, and 20) after treatment with LPS and TGF-ß for 24 h, as indicated. RNA was separated by gel electrophoresis, and Northern blot analysis was performed with probes specific for the VJ regions of the endogenous or the episomal Ig{kappa} gene.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A paused pol II at the Ig{kappa} gene in 70Z/3 cells

The mouse pre-B cell line 70Z/3 contains a functionally rearranged, nonexpressed Ig{kappa} gene that is transcriptionally activated between 1 and 6 h after stimulation with LPS (Fig. 1Go, lanes 1–5) (1). The transcriptional activation of the Ig{kappa} gene by LPS is under the negative control of TGF-ß (lanes 11–15) (34). The exact transcription start site of the Ig{kappa} gene in 70Z/3 cells was mapped in a primer extension experiment. In the presence of LPS, specific fragments were detected that do not appear in untreated cells (Fig. 2GoA, lanes 3 and 4), showing that the transcription start site is located about 30 bp upstream of the translation start codon.



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  		FIGURE 1. Transcriptional regulation of the Ig{kappa} gene in 70Z/3 cells. Cells were cultivated in the absence or presence of LPS and TGF-ß. Total cellular RNA was prepared at the indicated time points and separated by gel electrophoresis, and Northern blot analysis was performed using the Ig{kappa} gene C region as a probe.

 
We employed two methods to determine whether a paused pol II is present at the Ig{kappa} gene. First, an in vivo footprint experiment using ligation-mediated PCR was performed to map single-stranded regions downstream of the Ig{kappa} gene promoter (31, 32). The presence of a paused pol II should result in a transcription bubble that contains a region of unpaired DNA. Unpaired T residues in a transcription bubble are highly reactive to oxidation with potassium permanganate (KMnO4) (21, 35). Compared with purified genomic DNA treated with KMnO4 (Fig. 2GoB, lane 2), DNA treated with KMnO4 in intact nuclei displayed hypersensitive T residues in the sense strand at positions +1, +2, +6, +7, +19, +38, +52, +58, +64, and +67 (lane 3). A pronounced hypersensitivity was observed for the region between 38 and 67 bp downstream of the transcription start site. Pretreatment of 70Z/3 cells with LPS resulted in the same pattern of hypersensitive T residues (lane 4).

Single-stranded regions in DNA can be caused by a paused transcription complex, but also by binding of other protein complexes. To test whether the unpaired T residues represent transcription bubbles, a high resolution nuclear run-on experiment was performed (22). In a run-on experiment, paused pol II complexes and actively transcribing pol II, which were stalled during preparation of the nuclei, become activated by addition of high concentrations of nucleotides and transcribe a short stretch of RNA, thus revealing the distribution of pol II complexes along a gene. Labeled nuclear run-on RNA was hybridized to a set of seven 50-nucleotide-long oligonucleotides Up and A–F (Fig. 2GoC), corresponding to the promoter-proximal region of the Ig{kappa} gene in 70Z/3 cells. The filters were subjected to a RNase A-containing wash step after the hybridization to prevent unspecific background on the filters and to assure that no labeled RNA from neighboring sequences contributes as an overhang to a hybridization signal.

A run-on signal was obtained for oligonucleotide B spanning nucleotides +51 to +100, while signals for oligonucleotides upstream and downstream of oligonucleotide B were not detectable (Fig. 3GoA, lane 1). In contrast, no run-on signal for oligonucleotide B was observed for mouse erythroleukemia cells that do not contain a rearranged Ig{kappa} gene (lane 2). Signals obtained with a homogenously labeled Ig{kappa} RNA transcribed by T7 RNA polymerase served as a control for hybridization efficiency (lane 3). The run-on signal for oligonucleotide B in 70Z/3 cells and its absence in mouse erythroleukemia cells indicate that a paused pol II is present downstream of the RNA start site of the rearranged Ig{kappa} gene promoter. Treatment of 70Z/3 cells with LPS for 4 h resulted in an increase of hybridization signals for oligonucleotides A and C–F (Fig. 3GoB, lanes 4–6). This indicates that NF-{kappa}B activation confers processivity to the paused pol II, and that reinitiation of pol II is stimulated. The intensity of the run-on signal corresponding to oligonucleotide B was not affected by LPS. This correlates with the results from the footprint experiment showing the same pattern of hypersensitive T residues in nuclei of untreated and LPS-treated cells, indicating that a paused pol II is present downstream of the transcription initiation site of the Ig{kappa} gene, irrespective of whether the gene is transcribed.



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  		FIGURE 3. Pol II distribution within promoter-proximal sequences of the Ig{kappa} gene. A, B, and C, Nuclear run-on experiment with 70Z/3 and mouse erythroleukemia (MEL) cells. Nuclei were isolated at the indicated time points after treatment of the cells with or without LPS and TGF-ß, and run-on reactions were performed by allowing transcription in presence of [{alpha}-32P]CTP. Labeled RNAs were hybridized to a set of seven antisense oligonucleotides Up and A–F comprising the region immediately upstream and downstream of the transcription start site of the Ig{kappa} gene. The 7SK oligonucleotide served as a pol III transcription control. A, Lane 3, Control for hybridization efficiency. Uniformly labeled RNA of the Ig{kappa} gene transcribed in vitro by T7 RNA polymerase was hybridized to the run-on oligonucleotides Up and A–F.

 
Because TGF-ß inhibits the transcriptional activation of the Ig{kappa} gene by LPS, we tested whether TGF-ß interferes with pol II initiation and promoter-proximal pausing. Nuclear run-on experiments showed that the run-on signal for oligonucleotide B (Fig. 3GoC, lane 1) was not affected by TGF-ß (lanes 2–5). When 70Z/3 cells were induced with LPS in presence of TGF-ß, no increase of hybridization signals was observed for oligonucleotides A and C–F (lane 6). This indicates that TGF-ß does not interfere with initiation and pausing of pol II, but inhibits pol II processivity.

Regulation of the episomal Ig{kappa} gene by NF-{kappa}B and TGF-ß

To study pausing and activation of pol II at the Ig{kappa} gene promoter in more detail, the regulation of the Ig{kappa} gene was reconstituted on DNA constructs. In general, the expression of integrated constructs can be influenced by the chromosomal context, as has also been demonstrated for Ig{kappa} gene constructs (11). To circumvent position effects, the Ig{kappa} gene was introduced into 70Z/3 cells on an episomal vector. Bovine papilloma virus (BPV) was chosen, because it replicates episomally in mouse cells and is maintained with a specific copy number (36). We constructed pBPV-Ig{kappa} containing a genomic, rearranged copy of the Ig{kappa} gene from a mouse myeloma cell line. In addition, pBPV-Ig{kappa}{Delta} containing only the Ig{kappa} gene promoter and VJ region, but not the {kappa}Ei/MAR and C region, was constructed (Fig. 4GoA). Stable cell lines were established and the episomal status of the constructs was confirmed by Southern blot analysis. The plasmids were maintained with approximately one copy per cell (data not shown).

To study whether the expression of the episomal Ig{kappa} gene was regulated by LPS and TGF-ß, Northern blot analysis was performed. Transcripts derived from the endogenous and the episomal Ig{kappa} gene can be distinguished by using hybridization probes specific for the respective VJ regions. The endogenous Ig{kappa} gene was up-regulated by LPS in 70Z/3 cells and also in the cell lines containing pBPV, pBPV-Ig{kappa}, or pBPV-Ig{kappa}{Delta} (Fig. 4GoB, lanes 2, 4, 6, and 10), indicating that the transfection and selection procedures did not affect the LPS signaling pathway. In parallel, RNA was analyzed with a probe specific for the episomal Ig{kappa} gene. 70Z/3 cells and cell lines transfected with pBPV or pBPV-Ig{kappa}{Delta} did not show transcripts of the episomal Ig{kappa} gene (lanes 12, 14, and 20). Only the cell line carrying pBPV-Ig{kappa} with the full-length Ig{kappa} gene showed activation of Ig{kappa} gene transcription by LPS (lane 16). This activation was suppressed by TGF-ß (lane 18). Thus, the introduced episomal Ig{kappa} gene is regulated by LPS and TGF-ß, like the endogenous Ig{kappa} gene. Notably, cells carrying pBPV-Ig{kappa} showed low level expression of the episomal Ig{kappa} gene, even in the absence of LPS (lane 15). This expression was resistant to TGF-ß (lane 17), indicating that TGF-ß specifically affected NF-{kappa}B-mediated activation, but not basal transcription of the Ig{kappa} gene.

Formation of a paused pol II at the episomal Ig{kappa} gene, but not at Ig{kappa}{Delta}

To investigate whether the episomal Ig{kappa} gene establishes a paused pol II, nuclear run-on experiments were performed with oligonucleotides specific for the promoter-proximal region of the episomal Ig{kappa} gene (Fig. 5GoA). The cell line carrying pBPV-Ig{kappa} displayed a strong run-on signal corresponding to oligonucleotide A' (Fig. 5GoB, lane 1) compared with signals obtained with a homogenously labeled control RNA (Fig. 5GoC). This revealed a high density of pol II on oligonucleotide A'. Because this oligonucleotide spans the region from nucleotides +1 to +50, the position of the paused pol II at the episomal Ig{kappa} gene appears to be more proximal to the promoter compared with the observed pause site at the endogenous Ig{kappa} gene in 70Z/3 cells.

The core promoter region and upstream regulatory elements are sufficient for initiation and pausing of pol II at the hsp70 and the c-myc genes (21, 37). The construct pBPV-Ig{kappa}{Delta} contains the promoter and VJ region of Ig{kappa}, but not the {kappa}Ei/MAR and the C region. In contrast to pBPV-Ig{kappa}-transfected cells, run-on experiments with nuclei derived from pBPV-Ig{kappa}{Delta}-transfected cell lines did not show a detectable signal for oligonucleotide A' (Fig. 5GoB, lane 2). This indicates that formation of a paused pol II at the episomal Ig{kappa} gene promoter requires additional sequences that are contained in Ig{kappa}, but not in Ig{kappa}{Delta}.

Pausing of pol II at the Ig{kappa} gene in human B cells

To see whether pausing of pol II at the Ig{kappa} gene is conserved in other cell lines, we used the mature human B cell line Raji that has been isolated from a Burkitt lymphoma patient (38). In contrast to the pre-B cell line 70Z/3, Raji cells contain high constitutive NF-{kappa}B activity (data not shown), as expected for mature B cells. For transfection into Raji cells, we used an episomal vector system for human B cells based on EBV (39). Ig{kappa} and Ig{kappa}{Delta} fragments (Fig. 4GoA) were inserted into pEBV, resulting in pEBV-Ig{kappa} and pEBV-Ig{kappa}{Delta}. Consistent with constitutive NF-{kappa}B activity, the episomal Ig{kappa} gene was highly expressed in Raji cells transfected with pEBV-Ig{kappa} (Fig. 6GoA, lane 3). Transcripts of the episomal Ig{kappa} gene could not be detected in pEBV (lane 1)- or pEBV-Ig{kappa}{Delta} (lane 2)-transfected cell lines.



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  		FIGURE 6. Pausing of pol II at the episomal Ig{kappa} gene in Raji cells. A, Transcriptional regulation of the episomal Ig{kappa} gene in Raji cells. Cells were transfected with EBV-based constructs, and stable cell lines were established. Total cellular RNA was prepared from cells containing pEBV (lane 1), pEBV-Ig{kappa}{Delta} (lane 2), and pEBV-Ig{kappa} (lanes 3–14). RNA isolated from untreated cells and cells cultivated in presence of cyclosporin A for the indicated times was separated by gel electrophoresis. Northern blot analysis was performed using a probe specific for the VJ region of the episomal Ig{kappa} gene. B, Nuclear run-on experiment with established Raji cell lines. Nuclei from cell lines containing pEBV-Ig{kappa} (lanes 1 and 2) and pEBV-Ig{kappa}{Delta} (lane 3) were isolated after treatment with cyclosporin A for 24 h, if indicated, and run-on reactions were performed. Labeled RNAs were hybridized to a set of five antisense oligonucleotides, Up' and A'–D', comprising the region immediately upstream and downstream of the transcription start site of the episomal Ig{kappa} gene. The weak hybridization signal for oligonucleotide D' in lane 3 has not been observed in repeated experiments. The 7SK oligonucleotide served as a pol III transcription control.

 
To modulate the constitutive NF-{kappa}B activity in Raji cells, we used the potent immunosuppressive agent cyclosporin A. Cyclosporin A has been shown to inhibit the nuclear translocation and the in vitro DNA binding of NF-{kappa}B in monocytes/macrophages and T cells (40, 41) by preventing the inactivation of I{kappa}B (42, 43). The down-regulation of episomal Ig{kappa} RNA levels by cyclosporin A in Raji cells (lanes 4–8) confirmed that NF-{kappa}B activity is necessary for Ig{kappa} gene expression. Thus, the transcriptional regulation of the Ig{kappa} gene in Raji cells by NF-{kappa}B is comparable with the regulation observed in 70Z/3 cells.

To investigate whether treatment with cyclosporin A results in inhibition of processive transcription by pausing of pol II at the episomal Ig{kappa} gene, nuclear run-on experiments were performed. Run-on signals appeared for oligonucleotides A'–D' in pEBV-Ig{kappa}-transfected cells (Fig. 6GoB, lane 1), which correlates with the high mRNA level expressed from pEBV-Ig{kappa}. Treatment of cells with cyclosporin A resulted in a strong reduction of signals corresponding to oligonucleotides B'–D', whereas oligonucleotide A' still maintained a high run-on signal (lane 2). This indicates that inhibition of NF-{kappa}B activity by cyclosporin A resulted in pausing of pol II proximal to the Ig{kappa} gene promoter in Raji cells. The cell line transfected with pEBV-Ig{kappa}{Delta} showed no run-on signal for oligonucleotide A' (lane 3), confirming the observation in 70Z/3 cells that the promoter region of the Ig{kappa} gene is not sufficient to establish a paused pol II.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Initiation and pausing of pol II at the Ig{kappa} gene

This study investigated whether regulation of Ig{kappa} gene expression involves promoter-proximal pausing of pol II. In nuclear run-on experiments, a hybridization signal for a DNA probe complementary to the region between 51 and 100 nucleotides downstream of the transcription initiation site was observed in 70Z/3 cells, while no signals for DNA probes complementary to regions upstream and downstream thereof were obtained in absence of Ig{kappa} gene expression. For labeling of the run-on RNA, [{alpha}-32P]CTP was used. Because no hybridization signal for oligonucleotide A was observed, the catalytic site of pol II seems to be located downstream of bp +45 (the last C residue in the sequence complementary to oligonucleotide A). To generate the run-on signal on oligonucleotide B, at least one labeled CTP must have been incorporated in the run-on RNA. Therefore, the catalytic site of pol II seems to reside upstream of bp +90 (the last C residue in the sequence complementary to oligonucleotide B). The nuclear run-on transcription is restricted to a short promoter-proximal region and might be inhibited by obstacles further downstream, e.g., nucleosomes.

In in vivo footprint experiments, a pronounced hypersensitivity of T residues toward a single strand-specific probe was observed in the region from bp +38 to +67. This coincides well with the assumption that the catalytic site of the paused pol II is located between bp +45 and +90. A paused pol II complex was detectable, irrespective of whether the Ig{kappa} gene was repressed or transcribed, suggesting that the pause site is reoccupied immediately after activation of the stalled pol II. This is similar to other genes that have been found to be regulated at the level of RNA elongation by promoter-proximal pausing of pol II (44, 45).

To study initiation and pausing of pol II, we reconstituted Ig{kappa} gene regulation on stably transfected episomal constructs in a mouse pre-B and a mature human B cell line. Like the endogenous Ig{kappa} gene in 70Z/3 cells, the episomal Ig{kappa} genes in 70Z/3 and Raji cells established a paused pol II downstream of the transcription start site. In addition, the transcription of the episomal Ig{kappa} gene was NF-{kappa}B dependent and inhibited by TGF-ß, indicating that major features of regulation of Ig{kappa} gene transcription were reconstituted on episomes. In comparison with the chromosomal Ig{kappa} gene, the pause site of pol II on the episomes appeared to be located more promoter proximal. Variations in the position of pause sites have previously been noticed for the c-myc P2 promoter, suggesting that mechanisms contributing to the phenomenon of pausing may not strictly depend on the sequence at the pause site (46). Sequence-independent pausing of pol II can also be observed at hsp70/yp1 fusion constructs. The hsp70 gene in Drosophila melanogaster harbors a promoter-proximal paused pol II, whereas no paused pol II is established at the yp1 gene. In transgenic flies, sequences that reside upstream of the hsp70 TATA box, when fused upstream of the yp1 TATA box, specify the formation of a paused pol II on the 5' end of this hybrid gene (37). This is comparable with the observation made in this study. The endogenous and the episomal Ig{kappa} genes both contain an octamer site and a TATA element upstream of the transcription initiation site, but no sequence similarity exists for the pause sites of pol II at these genes.

The construct Ig{kappa}{Delta} containing the Ig{kappa} gene promoter, the VJ region, and part of the intron was not sufficient for initiation and pausing of pol II on episomal constructs. Because the full-length construct containing the {kappa}Ei/MAR and C region was sufficient for initiation and pausing of pol II, this suggests that elements within these sequences may interact with the promoter even if the Ig{kappa} gene is not transcribed. The episomal constructs used in this study will be instrumental to determine which elements within the deleted fragment are required for initiation and pausing of pol II.

The Ei/MAR element in the Ig heavy chain plays an important role for promoter accessibility (16). If DNA constructs containing the T7 RNA polymerase promoter together with and without the Ei/MAR of the heavy chain gene were inserted into transgenic mice, transcription experiments in isolated nuclei revealed that the Ei/MAR element was indispensable to allow T7 RNA polymerase transcription. Our observation suggests that the {kappa}Ei/MAR might function in a similar way to allow initiation and pausing of pol II, as Ei/MAR of the Ig heavy chain gene does in allowing accessibility for a heterologous transcription complex (16).

Regulation of pol II processivity by NF-{kappa}B and TGF-ß

The transcriptionally engaged pol II was observed at the Ig{kappa} gene promoter in 70Z/3 cells in the absence of NF-{kappa}B activity. Treatment of 70Z/3 cells with LPS increased the transcription rate in the Ig{kappa} gene region downstream of the pause site and induced high levels of Ig{kappa}-specific mRNA. LPS activates several signaling pathways involving tyrosine kinases and mitogen-activated protein kinases (47). However, for Ig{kappa} gene activation, the NF-{kappa}B pathway seems to be necessary (5). Other LPS-induced binding activities within the {kappa}Ei contribute only minor to enhancer activity (48, 49, 50). Therefore, it appears very likely that NF-{kappa}B is involved in activation of pol II at the pause site. In support of this notion, the inhibition of NF-{kappa}B activity in Raji cells by cyclosporin A decreased the transcription rate downstream of the pause site without a significant effect on pausing of pol II. How could NF-{kappa}B mediate the activation of the paused pol II? Several studies suggest that phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of pol II plays an important role in the activation of promoter-proximal paused pol II. The hypophosphorylated CTD is associated with initiation at the promoter and pausing, whereas hyperphosphorylation of CTD corresponds to an actively transcribing pol II (51, 52). The viral transactivator protein Tat confers processivity to the pol II paused downstream of the HIV-1 long terminal repeat promoter. Tat has recently been shown to stimulate phosphorylation of the CTD by binding to cyclinT/cdk9 (53, 54). It is possible that binding of NF-{kappa}B to the {kappa}Ei results in recruitment or activation of kinases, thereby inducing pol II processivity. It is also possible that NF-{kappa}B recruits other activities to the transcription machinery such as a histone acetyltransferase. For the paused pol II at an episomal c-myc gene, it has been shown that inhibitors of histone deacetylases induce processivity (55). Also, constitutive transcription of an episomal c-myc gene by insertion of an Ig enhancer element is accompanied by hyperacetylation of histones along the episomal gene construct (56). Therefore, it could be of importance that NF-{kappa}B had been previously shown to interact with the histone acetyltransferase p300/CBP (57, 58, 59). It is possible that NF-{kappa}B affects also the initiation rate at the Ig{kappa} gene promoter. At heat-shock genes, it has been observed with in vitro reconstituted chromatin templates that the transcription factor HSF does stimulate both processivity of the paused pol II complex and reinitiation of pol II at following rounds of transcription (60). At later stages in B cell differentiation, the {kappa}Ei is dispensable for Ig{kappa} gene transcription and becomes replaced by the {kappa}E3' (10, 13), suggesting that factors other than NF-{kappa}B can control pol II processivity.

TGF-ß is able to overcome NF-{kappa}B-mediated transcriptional activation of the Ig{kappa} gene. Gel-shift experiments showed that TGF-ß does not inhibit the activation and binding of NF-{kappa}B to the {kappa}Ei (61, 62). Therefore, two possibilities emerged how TGF-ß could inhibit NF-{kappa}B-mediated Ig{kappa} gene transcription: 1) TGF-ß could inhibit initiation and pausing of pol II, or 2) it could prevent processive transcription. Our finding that TGF-ß did not influence the amount of pol II paused at the Ig{kappa} gene suggests that TGF-ß might interfere with a late step in Ig{kappa} gene activation. For example, it may block a signal between the enhancer-bound NF-{kappa}B and the paused pol II. Consistently, IFN-{gamma} activates Ig{kappa} gene transcription in 70Z/3 cells in a NF-{kappa}B-independent and TGF-ß-resistant manner (62). This suggests that two alternative signaling pathways converge at the paused pol II. A model for activation of Ig{kappa} gene transcription is shown in Fig. 7Go.



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  		FIGURE 7. Schematic illustration of Ig{kappa} gene activation. A, A deletion construct without the {kappa}Ei/MAR and C region does not establish a promoter-proximal paused pol II. B, A pol II complex initiates and pauses at the Ig{kappa} gene in absence of NF-{kappa}B activity. Processive transcription at the Ig{kappa} gene is induced upon binding of NF-{kappa}B to its consensus site within the {kappa}Ei/MAR. TGF-ß inhibits processivity of pol II.

 
The pause site of pol II resides upstream of the 5' boundary of somatic hypermutation

Somatic hypermutation occurs at Ig genes during B cell differentiation and generates the secondary Ab repertoire by introducing nucleotide substitutions along the VJ region. The rate of mutations sharply rises about 150 bp downstream of the transcription start site, whereas the distal border is not well defined (63, 64).

Several studies have linked somatic hypermutation to transcription (65, 66). The observation of a paused pol II provides a possible explanation for the finding that the 5' boundary of somatic hypermutation lies downstream of the transcriptional start site in the leader intron. Pausing of pol II at the Ig{kappa} gene might be of functional relevance for induction of the hypermutation process, and the hypermutator might only work after pol II has passed the pause site. As described above, pol II at the pause site is activated by CTD hyperphosphorylation. The hyper- but not the hypophosphorylated form of CTD is capable of binding and recruiting various enzymatic activities to the transcriptional machinery. This includes the enzymes for mRNA 5' capping, splicing, as well as mRNA 3' end formation (67, 68, 69). Thus, a large complex may exist that carries out coupled transcription, splicing, and cleavage polyadenylation of mRNA precursors if processive transcription is induced (53). It is possible that this complex also recruits factors for hypermutation. Alternatively, a factor for hypermutation could also be loaded during initiation, but remains inactive until pol II switches into the processive transcription mode. A critical role for the {kappa}Ei in loading such a factor for hypermutation has been postulated before (65, 70).


   Acknowledgments
 
We are grateful to R. Mocikat for providing 70Z/3 cells and a vector containing the Ig{kappa} gene clone T1. We thank D. A. Wolf and G. Klobeck, and R. Mocikat for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 190), the Deutsche Krebshilfe, the European Community, and the Fonds der Chemischen Industrie. Back

2 Current address: Laboratory for Physiological Chemistry, Utrecht University, Utrecht, The Netherlands. Back

3 Address correspondence and reprint requests to Dr. Dirk Eick, GSF, Institute for Clinical Molecular Biology and Tumor Genetics, Marchioninistrasse 25, D-81377 Munich, Germany. E-mail address: Back

4 Abbreviations used in this paper: MAR, matrix attachment region; BPV, bovine papilloma virus; CTD, carboxyl-terminal domain; {kappa}Ei, Ig{kappa} gene intron enhancer; pol, polymerase. Back

Received for publication May 25, 1999. Accepted for publication July 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Paige, C. J., P. W. Kincade, P. Ralph. 1978. Murine B cell leukemia line with inducible surface immunoglobulin expression. J. Immunol. 121:641.[Abstract/Free Full Text]
  2. Wall, R., M. Kuehl. 1983. Biosynthesis and regulation of immunoglobulins. Annu. Rev. Immunol. 1:393.[Medline]
  3. Maki, R., J. Kearney, C. Paige, S. Tonegawa. 1980. Immunoglobulin gene rearrangement in immature B cells. Science 209:1366.[Abstract/Free Full Text]
  4. Sen, R., D. Baltimore. 1986. Inducibility of {kappa} immunoglobulin enhancer-binding protein NF-{kappa}B by a posttranslational mechanism. Cell 47:921.[Medline]
  5. Lenardo, M., J. W. Pierce, D. Baltimore. 1987. Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility. Science 236:1573.[Abstract/Free Full Text]
  6. Queen, C., D. Baltimore. 1983. Immunoglobulin gene transcription is activated by downstream sequence elements. Cell 33:741.[Medline]
  7. Picard, D., W. Schaffner. 1984. A lymphocyte-specific enhancer in the mouse immunoglobulin {kappa} gene. Nature 307:80.[Medline]
  8. Staudt, L. M., M. J. Lenardo. 1991. Immunoglobulin gene transcription. Annu. Rev. Immunol. 9:373.[Medline]
  9. Cockerill, P. N., W. T. Garrard. 1986. Chromosomal loop anchorage of the {kappa} immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44:273.[Medline]
  10. Meyer, K. B., M. S. Neuberger. 1989. The immunoglobulin {kappa} locus contains a second, stronger B-cell-specific enhancer which is located downstream of the constant region. EMBO J. 8:1959.[Medline]
  11. Blasquez, V. C., M. Xu, S. C. Moses, W. T. Garrard. 1989. Immunoglobulin {kappa} gene expression after stable integration. I. Role of the intronic MAR and enhancer in plasmacytoma cells. J. Biol. Chem. 264:21183.[Abstract/Free Full Text]
  12. Blasquez, V. C., M. A. Hale, K. W. Trevorrow, W. T. Garrard. 1992. Immunoglobulin {kappa} gene enhancers synergistically activate gene expression but independently determine chromatin structure. J. Biol. Chem. 267:23888.[Abstract/Free Full Text]
  13. Pongubala, J. M., M. L. Atchison. 1991. Functional characterization of the developmentally controlled immunoglobulin {kappa} 3' enhancer: regulation by Id, a repressor of helix-loop-helix transcription factors. Mol. Cell. Biol. 11:1040.[Abstract/Free Full Text]
  14. Fulton, R., B. Van-Ness. 1993. {kappa} immunoglobulin promoters and enhancers display developmentally controlled interactions. Nucleic Acids Res. 21:4941.[Abstract/Free Full Text]
  15. Henderson, A. J., K. L. Calame. 1995. Lessons in transcriptional regulation learned from studies on immunoglobulin genes. Crit. Rev. Eukaryotic Gene Expression 5:255.[Medline]
  16. Jenuwein, T., W. C. Forrester, L. A. Fernandez-Herrero, G. Laible, M. Dull, R. Grosschedl. 1997. Extension of chromatin accessibility by nuclear matrix attachment regions. Nature 385:269.[Medline]
  17. Lichtenstein, M., G. Keini, H. Cedar, Y. Bergman. 1994. B cell-specific demethylation: a novel role for the intronic {kappa} chain enhancer sequence. Cell 76:913.[Medline]
  18. Takeda, S., Y. R. Zou, H. Bluethmann, D. Kitamura, U. Muller, K. Rajewsky. 1993. Deletion of the immunoglobulin {kappa} chain intron enhancer abolishes {kappa} chain gene rearrangement in cis but not {lambda} chain gene rearrangement in trans. EMBO J. 12:2329.[Medline]
  19. Hiramatsu, R., K. Akagi, M. Matsuoka, K. Sakumi, H. Nakamura, L. Kingsbury, C. David, R. R. Hardy, K. Yamamura, H. Sakano. 1995. The 3' enhancer region determines the B/T specificity and pro-B/pre-B specificity of immunoglobulin V{kappa}-J{kappa} joining. Cell 83:1113.[Medline]
  20. Wagner, S. D., M. S. Neuberger. 1996. Somatic hypermutation of immunoglobulin genes. Annu. Rev. Immunol. 14:441.[Medline]
  21. Krumm, A., T. Meulia, M. Brunvand, M. Groudine. 1992. The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. Genes Dev. 6:2201.[Abstract/Free Full Text]
  22. Strobl, L. J., D. Eick. 1992. Holdback of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo. EMBO J. 11:3307.[Medline]
  23. Lis, J., C. Wu. 1993. Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell 74:1.[Medline]
  24. Plet, A., D. Eick, J. M. Blanchard. 1995. Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns. Oncogene 10:319.[Medline]
  25. Yankulov, K., J. Blau, T. Purton, S. Roberts, D. L. Bentley. 1994. Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell 77:749.[Medline]
  26. Croce, C. M.. 1993. Molecular biology of lymphomas. Semin. Oncol. 20:31.[Medline]
  27. Strobl, L. J., F. Kohlhuber, J. Mautner, A. Polack, D. Eick. 1993. Absence of a paused transcription complex from the c-myc P2 promoter of the translocation chromosome in Burkitt’s lymphoma cells: implication for the c-myc P1/P2 promoter shift. Oncogene 8:1437.[Medline]
  28. Hortnagel, K., J. Mautner, L. J. Strobl, D. A. Wolf, B. Christoph, C. Geltinger, A. Polack. 1995. The role of immunoglobulin {kappa} elements in c-myc activation. Oncogene 10:1393.[Medline]
  29. Parslow, T. G., D. L. Blair, W. J. Murphy, D. K. Granner. 1984. Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence. Proc. Natl. Acad. Sci. USA 81:2650.[Abstract/Free Full Text]
  30. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  31. Garrity, P. A., B. J. Wold. 1992. Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc. Natl. Acad. Sci. USA 89:1021.[Abstract/Free Full Text]
  32. Mueller, P. R., B. Wold. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780.[Abstract/Free Full Text]
  33. Altenburger, W., M. Steinmetz, H. G. Zachau. 1980. Functional and non-functional joining in immunoglobulin light chain genes of a mouse myeloma. Nature 287:603.[Medline]
  34. Lee, G., L. R. Ellingsworth, S. Gillis, R. Wall, P. W. Kincade. 1987. ß transforming growth factors are potential regulators of B lymphopoiesis. J. Exp. Med. 166:1290.[Abstract/Free Full Text]
  35. Wang, W., M. Carey, J. D. Gralla. 1992. Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. Science 255:450.[Abstract/Free Full Text]
  36. Mecsas, J., B. Sugden. 1987. Replication of plasmids derived from bovine papilloma virus type 1 and Epstein-Barr virus in cells in culture. Annu. Rev. Cell Biol. 3:87.
  37. Lee, H., K. W. Kraus, M. F. Wolfner, J. T. Lis. 1992. DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev. 6:284.[Abstract/Free Full Text]
  38. Epstein, M. A., B. G. Achong, Y. M. Barr, B. Zajac, G. Henle, W. Henle. 1966. Morphological and virological investigations on cultured Burkitt tumor lymphoblasts (strain Raji). J. Natl. Cancer Inst. 37:547.
  39. Yates, J. L., N. Warren, B. Sugden. 1985. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313:812.[Medline]
  40. Marienfeld, R., M. Neumann, S. Chuvpilo, C. Escher, B. Kneitz, A. Avots, A. Schimpl, E. Serfling. 1997. Cyclosporin A interferes with the inducible degradation of NF-{kappa}B inhibitors, but not with the processing of p105/NF-{kappa}B1 in T cells. Eur. J. Immunol. 27:1601.[Medline]
  41. Holschermann, H., F. Durfeld, U. Maus, A. Bierhaus, K. Heidinger, J. Lohmeyer, P. P. Nawroth, H. Tillmanns, W. Haberbosch. 1996. Cyclosporine A inhibits tissue factor expression in monocytes/macrophages. Blood 88:3837.[Abstract/Free Full Text]
  42. Frantz, B., E. C. Nordby, G. Bren, N. Steffan, C. V. Paya, R. L. Kincaid, M. J. Tocci, S. J. O’Keefe, E. A. O’Neill. 1994. Calcineurin acts in synergy with PMA to inactivate I {kappa}B/MAD3, an inhibitor of NF-{kappa}B. EMBO J. 13:861.[Medline]
  43. Meyer, S., N. G. Kohler, A. Joly. 1997. Cyclosporine A is an uncompetitive inhibitor of proteasome activity and prevents NF-{kappa}B activation. FEBS Lett. 413:354.[Medline]
  44. Bentley, D. L.. 1995. Regulation of transcriptional elongation by RNA polymerase II. Curr. Opin. Genet. Dev. 5:210.[Medline]
  45. Giardina, C., M. Perez Riba, J. T. Lis. 1992. Promoter melting and TFIID complexes on Drosophila genes in vivo. Genes Dev. 6:2190.[Abstract/Free Full Text]
  46. Wolf, D. A., L. J. Strobl, A. Pullner, D. Eick. 1995. Variable pause positions of RNA polymerase II lie proximal to the c-myc promoter irrespective of transcriptional activity. Nucleic Acids Res. 23:3373.[Abstract/Free Full Text]
  47. Sweet, M. J., D. A. Hume. 1996. Endotoxin signal transduction in macrophages. J. Leukocyte Biol. 60:8.[Abstract]
  48. Nelms, K., R. Hromas, B. Van Ness. 1990. Identification of a second inducible DNA-protein interaction in the {kappa} immunoglobulin enhancer. Nucleic Acids Res. 18:1037.[Abstract/Free Full Text]
  49. Nelms, K., B. Van Ness. 1990. Identification of an octamer-binding site in the human {kappa} light-chain enhancer. Mol. Cell. Biol. 10:3843.[Abstract/Free Full Text]
  50. Schanke, J. T., A. Marcuzzi, R. P. Podzorski, B. Van-Ness. 1994. An AP1 binding site upstream of the {kappa} immunoglobulin intron enhancer binds inducible factors and contributes to expression. Nucleic Acids Res. 22:5425.[Abstract/Free Full Text]
  51. O’Brien, T., S. Hardin, A. Greenleaf, J. T. Lis. 1994. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature 370:75.[Medline]
  52. Greenblatt, J.. 1997. RNA polymerase II holoenzyme and transcriptional regulation. Curr. Opin. Cell Biol. 9:310.[Medline]
  53. Zhu, Y., T. Pe’ery, J. Peng, Y. Ramanathan, N. Marshall, T. Marshall, B. Amendt, M. B. Mathews, D. H. Price. 1997. Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 11:2622.[Abstract/Free Full Text]
  54. Wei, P., M. E. Garber, S. M. Fang, W. H. Fischer, K. A. Jones. 1998. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451.[Medline]
  55. Albert, T., J. Mautner, J. O. Funk, K. Hortnagel, A. Pullner, D. Eick. 1997. Nucleosomal structures of c-myc promoters with transcriptionally engaged RNA polymerase II. Mol. Cell. Biol. 17:4363.[Abstract]
  56. Madisen, L., A. Krumm, T. R. Hebbes, M. Groudine. 1998. The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes. Mol. Cell. Biol. 18:6281.[Abstract/Free Full Text]
  57. Gerritsen, M. E., A. J. Williams, A. S. Neish, S. Moore, Y. Shi, T. Collins. 1997. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc. Natl. Acad. Sci. USA 94:2927.[Abstract/Free Full Text]
  58. Perkins, N. D., L. K. Felzien, J. C. Betts, K. Leung, D. H. Beach, G. J. Nabel. 1997. Regulation of NF-{kappa}B by cyclin-dependent kinases associated with the p300 coactivator. Science 275:523.[Abstract/Free Full Text]
  59. Zhong, H., R. E. Voll, S. Ghosh. 1998. Phosphorylation of NF-{kappa}B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell 1:661.[Medline]
  60. Sandaltzopoulos, R., P. B. Becker. 1998. Heat shock factor increases the reinitiation rate from potentiated chromatin templates. Mol. Cell. Biol. 18:361.[Abstract/Free Full Text]
  61. O’Brien, D. P., E. M. Oltz, B. G. Van Ness. 1997. Coordinate transcription and V(D)J recombination of the {kappa} immunoglobulin light-chain locus: NF-{kappa}B-dependent and -independent pathways of activation. Mol. Cell. Biol. 17:3477.[Abstract]
  62. Briskin, M., M. D. Kuwabara, D. S. Sigman, R. Wall. 1988. Induction of {kappa} transcription by interferon-{gamma} without activation of NF-{kappa}B. Science 242:1036.[Abstract/Free Full Text]
  63. Rogerson, B. J.. 1994. Mapping the upstream boundary of somatic mutations in rearranged immunoglobulin transgenes and endogenous genes. Mol. Immunol. 31:83.[Medline]
  64. Rada, C., A. Gonzalez Fernandez, J. M. Jarvis, C. Milstein. 1994. The 5' boundary of somatic hypermutation in a V{kappa} gene is in the leader intron. Eur. J. Immunol. 24:1453.[Medline]
  65. Neuberger, M. S., C. Milstein. 1995. Somatic hypermutation. Curr. Opin. Immunol. 7:248.[Medline]
  66. Peters, A., U. Storb. 1996. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4:57.[Medline]
  67. Mortillaro, M. J., B. J. Blencowe, X. Wei, H. Nakayasu, L. Du, S. L. Warren, P. A. Sharp, R. Berezney. 1996. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc. Natl. Acad. Sci. USA 93:8253.[Abstract/Free Full Text]
  68. McCracken, S., N. Fong, K. Yankulov, S. Ballantyne, G. Pan, J. Greenblatt, S. D. Patterson, M. Wickens, D. L. Bentley. 1997. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385:357.[Medline]
  69. McCracken, S., N. Fong, E. Rosonina, K. Yankulov, G. Brothers, D. Siderovski, A. Hessel, S. Foster, S. Shuman, D. L. Bentley. 1997. 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11:3306.[Abstract/Free Full Text]
  70. Goyenechea, B., N. Klix, J. Yelamos, G. T. Williams, A. Riddell, M. S. Neuberger, C. Milstein. 1997. Cells strongly expressing Ig({kappa}) transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers. EMBO J. 16:3987.[Medline]



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