©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activating Transcription Factor 1 and Cyclic AMP Response Element Modulator Can Modulate the Activity of the Immunoglobulin 3` Enhancer (*)

Jagan M. R. Pongubala (§) , Michael L. Atchison (¶)

From the (1) Department of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously we determined that the immunoglobulin kappa 3` enhancer (E3`) contains at least two functional DNA sequences (PU.1/NF-EM5 and E2A) within its 132-base pair active core. We have determined that the activities of these two sequences are insufficient to account for the entire activity of the 132-base pair core. Using site-directed linker scan mutagenesis across the core fragment we identified several additional functional sequences. We used one of these functional sequences to screen a gt11 cDNA expression library resulting in the isolation of cDNA clones encoding the transcription factors ATF-1 (activating transcription factor) and CREM (cyclic AMP response element modulator). Because ATF-1 and CREM are known to bind to cAMP response elements (CRE), this functional sequence was named the E3`-CRE. We show that dibutyryl cAMP can increase E3` enhancer activity, and in transient expression assays ATF-1 caused a 4-5-fold increase in the activity of the core enhancer while CREM- expression resulted in repression of enhancer activity. RNA analyses showed increased levels of ATF-1 mRNA during B cell development and some changes in CREM transcript processing. By joining various fragments of the E3` enhancer to the E3`-CRE, we observed that the E3`-CRE can synergistically increase transcription in association with the PU.1/NF-EM5 binding sites, suggesting a functional interaction between the proteins that bind to these DNA sequences. Consistent with this possibility, we found that ATF-1 and CREM can physically interact with PU.1. The isolation of activator and repressor proteins that bind to the E3`-CRE may relate to previous conflicting results concerning the role of the cAMP signal transduction pathway in gene transcription.


INTRODUCTION

Immunoglobulin light chain gene expression is under the control of two enhancers: the intron and the E3` enhancers. Both the intron and the 3` enhancers are inactive in Abelson virus-transformed pre-B cell lines in which the locus is silent. However, both enhancers are active at the B cell and plasma cell stages. Previously we determined that a 132-bp() centrally located segment within the E3` enhancer (the enhancer core) contains nearly the entire enhancer activity when assayed in plasmacytoma cells (1) . This same core segment is also active in pre-B cells, indicating the existence of flanking negative acting DNA sequences that repress enhancer activity at the pre-B cell stage (2) . Within the enhancer core we previously identified two functional DNA sequences. One sequence binds to the transcription factors PU.1 and NF-EM5, and the other binds to the transcription factor E2A (1, 2, 4) . The PU.1/NF-EM5 and E2A functional sequences were identified based on their enhancer activity when assayed as multimers (1) . Other functional sequences may exist in the core segment which are not active as multimers. These functional sequences may be revealed by a linker scan mutagenesis approach.

An interesting feature of gene transcription is its inducibility at the pre-B cell stage. Although the locus is transcriptionally silent in Abelson virus-transformed pre-B cells, certain agents can induce transcription. These agents include bacterial lipopolysaccharide, interleukin-1, and -interferon (5, 6, 7, 8, 9, 10) . Induction of transcription in some cases appears to require induction of the functional form of NF-B (8, 11, 12, 13, 14) . For instance, lipopolysaccharide treatment is believed to cause phosphorylation of IB resulting in its dissociation from NF-B. NF-B can then enter the nucleus where it can bind to the intron enhancer and initiate expression of the gene. Like the intron enhancer the E3` enhancer becomes active at the pre-B cell stage when cells are treated with lipopolysaccharide. However, the E3` enhancer does not require NF-B for its activity (1, 15) .

Although various agents can modulate locus expression, conflicting results have been reported. For instance, one report suggests that interleukin-1 and cAMP can increase transcription, whereas others suggest an inhibitory effect by the same compounds (7, 9, 16) . This issue may be clarified by identifying the nuclear factors that control locus transcription. In the studies presented here, we used linker scan mutagenesis to identify additional functional sequences within the E3` enhancer core. We show that one functional sequence binds to the leucine zipper transcription factors ATF-1 and CREM. Because these factors bind to cAMP response elements (CRE) we named this functional segment of the E3` enhancer the E3`-CRE. We show that cAMP levels can increase E3` enhancer activity, and ATF-1 can activate the E3` enhancer while CREM- can act as a repressor. We also show that the E3`-CRE can synergistically activate transcription in association with the PU.1/NF-EM5 binding sites. In addition, ATF-1 and CREM can physically interact with PU.1. These factors may provide a mechanism for the induction or repression of gene transcription in response to various agents.


MATERIALS AND METHODS

Plasmid Constructs

To prepare plasmid E3`coreTKCAT, the 132-bp AvaII- ApaLI core fragment of 3` enhancer was blunted by Klenow polymerase and cloned into the HincII site of pUC18. This fragment was excised by HindIII- BamHI digestion and ligated into the HindIII- BamHI sites of TKCAT containing sequences 109 to +51 of the herpesvirus thymidine kinase (TK) promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. PU.1+E2ATKCAT was prepared by cloning the E2A sequence with BamHI- BglII restriction sites at the BglII site adjacent to the PU.1/NF-EM5 sequence in a vector containing the liver, bone, kidney (LBK) alkaline phosphatase promoter (LBK44 vector kindly supplied by T. Kadesch, University of Pennsylvania). The PU.1/NF-EM5 and E2A sequence was excised by BamHI- BglII digestion and ligated into the BamHI site of TKCAT. Single copies of the PU.1/NF-EM5 and E2A sequences with BamHI and BglII ends were cloned into the BamHI site of TKCAT to yield constructs PU.1TKCAT and E2ATKCAT, respectively. Oligonucleotides containing the E3`-CRE+PU.1 or E3`-CRE+E2A sites were synthesized with BamHI and HindIII ends and cloned into the BamHI- HindIII sites of TKCAT to yield constructs E3`-CRE+PU.1TKCAT and E3`-CRE+E2ATKCAT, respectively. Linker scan mutants across the core fragment (A to M; except B) were created by a polymerase chain reaction (PCR) method (17) . For each mutation, divergent oligonucleotide primers with 10-bp overlaps (containing a new SalI restriction site) were used. The sequences of the mutant oligonucleotides used for generating the respective mutations are as follows. Linker scan mutant B was created by the method described by Kunkel (18) using a 40-bp synthetic oligonucleotide containing the mutant B sequences (5`-GTGTGACGGTAGCTAGCGTCGACCGTATCTTGGTCCATGG-3`). All mutations (A to M) were confirmed by DNA sequencing (19) . The PU.1-GST construct was prepared by inserting a PvuII- BamHI fragment containing the entire PU.1 coding sequence, 4 bp of 5`-untranslated sequence, and 337 bp of 3`-untranslated sequence by blunt end ligation in the blunted EcoRI site of pGEX-2TK (kindly provided by W. Kaelin, Dana Farber Cancer Institute).

Cell Culture and Transfection

S194 and 1-8 cells were grown and transfected as described previously (1) . Transfections generally contained 4 µg of test plasmid and 1 µg of the -galactosidase expression plasmid pCH110 (20) to normalize the transfection efficiency. For cotransfection experiments, 3-4 µg of reporter plasmid was cotransfected with various amounts of expression plasmid. The total concentration of DNA in each transfection was normalized with pUC18 plasmid DNA. Some transfected cells were incubated with BtcAMP (Sigma) for 16 h before harvest. Cell extracts were prepared by freeze-thawing and CAT assays and thin-layer chromatography were performed according to Gorman et al. (21) .

Electrophoretic Mobility Shift Assay (EMSA) and Methylation Interference Assay

Nuclear extracts were prepared by the method of Dignam et al. (22) , and EMSA was performed as described previously (1) . Antibody treatments were at room temperature for 20 min before addition of the probe. CREM antibodies were a gift from P. Sassone-Corsi. ATF-1 antibodies were obtained from Upstate Biotechnology Inc. CREB and c-fos antibodies were obtained from Santa Cruz Biotechnology Inc. For methylation interference assays, the E3`-CRE sequence with BamHI and BglII ends was cloned into the BamHI- BglII sites of the LBK44 vector. This plasmid was cut with either BamHI or BglII, dephosphorylated with calf intestine phosphatase, labeled with [-P]ATP by polynucleotide kinase, then digested with the appropriate second enzyme to release the oligonucleotide insert. Labeled DNA fragments were purified by polyacrylamide gel electrophoresis. DNA fragments were processed for dimethyl sulfate methylation interference footprinting with S194 nuclear extract as described previously (23) . Proteins made by in vitro translation were prepared from RNAs transcribed in vitro from full-length cDNAs with T7 RNA polymerase according to the manufacturer's specifications (Stratagene), and proteins were translated in vitro using nuclease-treated RNA-dependent rabbit reticulocyte lysates (Promega) at 30 °C for 60 min.

Screening the gt11 Library

A human HeLa cell-derived gt11 cDNA library (provided by P. Henthorn, University of Pennsylvania) was screened with a multimerized E3`-CRE probe according to the method of Vinson et al. (24) . The nucleotide sequence of the E3`-CRE probe is 5`-AGCAACTGTCAATAGCTACCGTCACA-3` (upper strand); 3`-TGTGACGGTAGCTATTGACAGTTGCT-5` (lower strand).

RNA Analyses

Poly(A)RNAs isolated from 3-1, 1-8, MPC11, or S194 cells were fractionated on 2.0% agarose-formaldehyde gels and transferred to Nytran (Schleicher & Schuell) according to the manufacturer's specifications. For reverse transcriptase PCR assays 1 µg of poly(A)RNA isolated from MPC11 plasmacytoma cells was copied into cDNA using primer B (5`-CATGCTGTAATCAGTTCATAG-3`) and reverse transcriptase in a 20-µl reaction containing 50 mM Tris-HCl (pH 8.3), 150 mM KCl, 10 mM MgCl, 15 mM dithiothreitol, and 0.5 mM dNTP at 42 °C for 1 h. Forty cycles of PCR were then performed with primer B and primer A (5`-GATTGAAGAAGAAAAATCAGA-3`) using the following cycle: 55 °C, 1 min; 72 °C, 2 min; 94 °C, 30 s. PCR products were cloned into plasmid pGEM-T (Promega) for DNA sequence analysis.

Preparation of GST-PU.1 Fusion Protein

The GST-PU.1 fusion protein was expressed in Escherichia coli BL21 (DE3) by induction with 1 mM isopropyl 1-thio--D-galactopyranoside (Sigma) for 2 h. Cells were harvested by centrifugation, resuspended in buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 (NETN buffer), and lysed by ultrasonication with 30-s pulses four times. Debris was pelleted by centrifugation (10,000 rpm, 30 min). The supernatant was mixed with glutathione-agarose (Sigma) and rocked at 4 °C for 1 h. Beads were then washed with NETN buffer to remove unbound proteins. An aliquot of GST-PU.1 linked to glutathione-agarose was assayed by SDS-polyacrylamide gel electrophoresis.

GST-PU.1 Affinity Chromatography

ATF-1 and CREM- proteins were prepared by in vitro transcription and translation in the presence of [S]methionine. The labeled ATF-1 and CREM- proteins were incubated with 20 µl of glutathione-agarose beads containing PU.1-GST or glutathione S-transferase alone for 1 h at 4 °C. After incubation, the beads were washed three times with NETN buffer, resuspended in SDS-polyacrylamide gel electrophoresis buffer, boiled, and analyzed by 10% SDS-polyacrylamide gel electrophoresis followed by autoradiography.


RESULTS

The PU.1/NF-EM5 and E2A Binding Sites Do Not Account for All of the Core Enhancer Activity

Previously, we determined that the sequences spanning nucleotide positions 391-523 of the 3` enhancer (numbering according to Ref. 25) contain most of the activity of the intact 1.1-kilobase enhancer (1) . Using nine multimerized 25-bp overlapping oligonucleotides spanning this core fragment we localized two functional regions. The first region binds to the transcription factors PU.1 and NF-EM5, and the second region binds to transcription factor E2A (1, 3, 4) . To test whether these two DNA segments account for the entire activity of the core fragment, we linked single copies of the PU.1/NF-EM5 plus E2A binding sites into a vector containing the thymidine kinase promoter driving expression of the CAT gene. Similarly, TKCAT constructs containing a single copy of either the PU.1/NF-EM5 or E2A binding sites were prepared. These constructs were transfected into S194 plasmacytoma cells, and CAT activities were measured. No significant CAT activity was observed with constructs containing single copies of either the PU.1/NF-EM5 or E2A DNA binding sites (Fig. 1). However, the construct containing both the PU.1/NF-EM5 and E2A binding sites exhibited 21% activity when compared with the activity of the entire core fragment. These results indicate that the PU.1/NF-EM5 and E2A binding sites can functionally synergize to yield enhancer activity. However, other DNA elements must be present in the core fragment which are necessary to yield 100% enhancer activity.


Figure 1: The PU.1/NF-EM5 and E2A binding sites do not account for all of the E3` enhancer activity. S194 cells were transfected with 4 µg of each DNA construct, and CAT activities were determined. The results of representative transfections are shown. Comparable results were obtained in at least two to three independent experiments. Above each lane is listed the transfected DNA construct, and the percent enhancer activity is indicated below each lane.



Linker Scan Mutations across the Core Fragment

To identify additional regulatory elements that are important for activity of the E3` enhancer, we prepared a series of 10-bp linker scan mutants (A to M; see Fig. 2) across the 132-bp core fragment. These constructs were individually transfected into 1-8 cells (a pre-B cell line) or S194 plasmacytoma cells, and CAT activities were determined (Fig. 3, A and B, respectively). It should be noted that although the entire E3` enhancer is inactive in pre-B cells, the 132-bp core fragment is active.


Figure 2: Linker scan mutations within the E3` enhancer core segment. The DNA sequences of the 10-bp linker scan mutants (A to M) across the core fragment of the E3` enhancer are shown. The wild type sequence is shown on the top line. Dashes represent nucleotide identities. Positions of the E3`-CRE, PU.1, NF-EM5, and E2A sequences are indicated.




Figure 3: Identification of new functional sequences in the E3` enhancer. 4 µg of each mutant construct was individually transfected into either panel A, 1-8 pre-B cells or panel B, S194 plasmacytoma cells, and CAT activities were determined. Below each lane is indicated the linker scan mutant used for transfection. CORE is the 132-bp E3` enhancer core cloned into TKCAT. Panel C, the relative activities of the linker scan mutants in 1-8 pre-B cells ( solid bars) and S194 plasmacytoma cells ( open bars) are plotted. The activity of the wild type core fragment was considered as 100%, and the activity of each mutant construct is calculated relative to this value. Data are from an average of two to three transfections. The relative activity for each construct varied by less than 10%.



Consistent with our previous results, mutation of the DNA sequences spanning the PU.1/NF-EM5 (mutations F, G, and H) and E2A (mutations I and J) binding sites greatly reduced enhancer activity in 1-8 and S194 cells (Fig. 3, A and B). Linker scan K also reduced enhancer activity in both cell types, but as yet no protein binding to this DNA sequence has been identified (1) . In addition, linker scans A, B, and D showed reduced enhancer activity. Linker scan B is particularly interesting because it reduced enhancer activity to near background levels in 1-8 pre-B cells but only reduced activity to 30% of wild type in S194 plasmacytoma cells (Fig. 3 C). We demonstrated previously that multimerized oligonucleotides containing wild type sequences of mutants A and B (oligonucleotide 1), mutants B and C (oligonucleotide 2), and mutants D and E (oligonucleotide 3) are inactive as enhancers when transfected into S194 plasmacytoma cells (1) . Therefore, these DNA segments cannot exhibit enhancer activity on their own and must functionally interact with other enhancer segments to be active. Our results here with the mouse E3` enhancer are consistent with a mutational analysis of the human 3` enhancer (26) . These studies showed that substitution of nucleotides in the human enhancer at sites similar to linker scans B and D reduced enhancer activity to 21 and 19%, respectively, in human Burkitt lymphoma cells (26) .

Identification of Protein-DNA Contact Sites

Previously we showed that oligonucleotide 2, which contains the entire wild type linker scan B and C sequences and 4 bp of linker scan D sequence (nucleotides 400-424), binds to factors present in nuclear extracts of pre-B, plasmacytoma, and 3T3 fibroblast cells (1) . These factors result in the appearance of three shifted complexes in EMSAs: a slowly migrating doublet (complexes 1 and 2), and a third faster migrating complex (complex 3; Fig. 4). All bound complexes were efficiently competed by adding excess unlabeled self-oligonucleotide (Fig. 4; lane 2) but not by a nonspecific oligonucleotide ( lane 3).


Figure 4: Protein binding ability of wild type and mutant DNA probes. Various 4-bp mutant oligonucleotides (M.1 through M.6) were labeled with P, and EMSAs (with low ionic strength buffer) were performed using 3-1 pre-B cell nuclear extract. Parallel reactions were carried out with the wild type oligonucleotide 2 sequence (see Fig. 5 B) as probe in the presence of unlabeled mutant oligonucleotide competitors (M.1-M.6), self-oligonucleotide ( SELF), or a nonspecific oligonucleotide ( NS). DNA-protein complexes were resolved by 4% nondenaturing gel electrophoresis. Above each lane is indicated the labeled probe and unlabeled competitor oligonucleotides. Arrows point to complexes 1, 2, and 3. The wild type and mutated oligonucleotide sequences are shown in the lower panel.



To identify the nucleotides responsible for binding to nuclear factors we prepared consecutive 4-bp mutations across the 25-bp oligonucleotide 2 sequence to yield six mutant oligonucleotides (M.1-M.6). Each mutant oligonucleotide was used as a labeled probe in EMSA (Fig. 4, lanes 4-9). Mutants M.1 and M.6 exhibited wild type binding patterns ( lanes 4 and 9). Mutant M.2 showed weak binding of complexes 1 and 2 but undetectable binding of complex 3 ( lane 5; a faint, faster migrating complex was observed with this probe as well as mutant M.3). Mutant M.3 was unable to yield any of the specific complexes ( lane 6). Mutants M.4 and M.5 completely abolished binding of complexes 1 and 2 but exhibited increased binding to the factor responsible for complex 3, perhaps because of the increased amount of probe available ( lanes 7 and 8).

Confirmatory results were obtained when these mutant oligonucleotides were used as unlabeled competitors ( lanes 10-15). Mutants M.1 and M.6 completely abolished factor binding to the wild type probe ( lanes 10 and 15). Mutant M.2 competed complexes 1 and 2 but not 3 ( lane 11), whereas mutant M.3 failed to compete any of the complexes ( lane 12). Finally, mutants M.4 and M.5 competed complex 3 but not complexes 1 and 2 ( lanes 13 and 14). These results suggest that the factor responsible for complex 3 binds between nucleotides 404 and 411, and the factors responsible for complexes 1 and 2 bind between nucleotides 404 and 419.

Dimethyl sulfate methylation interference assays were performed to define more narrowly the protein-DNA contact sites (Fig. 5 A). Footprints were generated with complexes 1 and 2, but unfortunately complex 3 did not yield sufficient material for methylation interference analysis. Studies with the upper DNA strand indicated that for complex 1, methylation of guanine residues 407 and 413 interfered with binding. On the bottom DNA strand methylation of guanines 409, 414, 417, and 418 inhibited complex 1 formation. Complex 2 exhibited partial contacts at guanines 409 and 414 in the lower strand and showed contacts at guanines 407 and 413 in the upper strand (Fig. 5 A). A summary of the protein DNA interaction studies is presented in Fig. 5B. Similar protein-DNA contact sites were observed by in vivo footprinting of the human light chain 3` enhancer (Fig. 5 C; 27).


Figure 5: Identification of protein-DNA contact sites. Panel A, the 25-bp oligonucleotide 2 sequence (see panel B) was used in dimethyl sulfate methylation interference assays. Lanes containing free ( F), complex 1 ( 1), or complex 2 ( 2) are indicated. The positions of the G residues that inhibited protein binding when methylated are indicated by arrows. Panel B, summary of the footprint and EMSAs. The oligonucleotide 2 sequence (nucleotides 400-424) is shown at the top, and the positions of linker scan mutants B, C, and D are indicated. Asterisks show protein contact sites identified by dimethyl sulfate methylation interference assays. Below are shown the CRE-like motifs in the sequence and the sequences of mutants M.1-M.6. The binding activity of each mutant as measured by EMSA is summarized in the lower right. Panel C, the protein contact sites of the mouse enhancer are compared with the contact sites of the human 3` enhancer determined by in vivo footprinting (27).



The above analyses indicate that protein contact sites within the oligonucleotide 2 sequence extend across the linker scan B and C sequences. EMSA complexes 1 and 2 result from factors that bind to sequences spanning linker scans B and C. On the contrary, EMSA complex 3 results from factors binding to the linker scan B region.

Isolation of cDNA Clones Encoding Proteins That Bind to Oligonucleotide 2

A gt11 cDNA expression library was screened with oligonucleotide 2 sequences (nucleotides 400-424) by the method of Vinson et al. (24) . After several rounds of screening, two strong positive cDNA clones were isolated. The DNA sequences of these clones indicated that they are identical to ATF-1 (27; identical to Tax response element binding protein, TREB36; 28) and CREM (29) . These proteins are known to bind to CREs. Inspection of the oligonucleotide 2 DNA sequence revealed two regions with homology to CRE-like sites (30; see Fig. 5B). We therefore named this segment of the 3` enhancer the E3`-CRE.

Binding of CREM and ATF-1 to the E3` Enhancer

EMSA with oligonucleotide 2 DNA sequences and CREM or ATF-1 protein prepared by in vitro transcription and translation yielded inconsistent results. Faint high mobility complexes were observed occasionally, suggesting that CREM and ATF-1 bind poorly on their own but may bind more efficiently in the presence of other nuclear proteins. To determine whether CREM or ATF-1 proteins were part of the EMSA complexes observed with oligonucleotide 2 sequences and nuclear extract proteins, we performed EMSAs with CREM and ATF-1 antibodies. We also included experiments with antibodies to two other proteins, CREB and c-fos, which bind to CRE-like sequences. Whereas preimmune, CREB, and c-fos antibodies had no effect on complexes 1, 2, and 3 (Fig. 6, lanes 2, 5, and 6), ATF-1 and CREM antibodies caused a loss of complexes 1 and 2 and the appearance of higher mobility complexes ( lanes 3 and 4). The above antibodies had no effect on the DNA binding of a distinct transcription factor, YY1 (data not shown). Therefore, ATF-1 and CREM, or closely related proteins, are associated with the formation of complexes 1 and 2.


Figure 6: ATF-1 and CREM bind to the E3`-CRE. Radiolabeled E3`-CRE oligonucleotide (oligonucleotide 2) was incubated with S194 nuclear extract proteins. Samples were either untreated ( lane 1), treated with preimmune sera ( lane 2), or treated with antibodies against CREM ( lane 3), ATF-1 ( lane 4), c-fos ( lane 5), or CREB ( lane 6).



We also performed mixing experiments with ATF-1 and CREM and nuclear extract proteins to determine the influence of ATF-1 and CREM on protein-DNA complex formation. For these experiments, various amounts of nuclear extract proteins were assayed alone or in combination with ATF-1 or CREM using the labeled E3`-CRE as probe. Mixing of 2 µl of ATF-1 prepared by in vitro translation with nuclear extract proteins caused a significant increase in the intensity of all bound complexes. Adding control rabbit reticulocyte lysate to the nuclear extract did not change the binding pattern (Fig. 7), nor did addition of CREM prepared by in vitro translation (data not shown). The above antibody and mixing experiments suggest that ATF-1 and CREM bind to DNA as a complex with other nuclear factors.


Figure 7: ATF-1 enhances the binding ability of nuclear factors to the E3`-CRE. Various amounts of nuclear extract proteins (from 3-1 pre-B cells) were assayed alone, in the presence of 2 µl of ATF-1 prepared by in vitro translation, or in the presence of control translation lysate ( RRL). The amount of nuclear extract protein and the presence of ATF-1 or control lysate ( RRL) are indicated above each lane. Arrows indicate the positions of complexes 1, 2, and 3.



cAMP Levels Can Influence E3` Enhancer Activity

Because CREM and ATF-1 are associated with the cAMP signal transduction pathway, we sought to determine whether increased cAMP levels could induce E3` enhancer activity in pre-B cells. One or two copies of the entire E3` enhancer were cloned into the TKCAT reporter plasmid. 1-8 pre-B cells were transfected with each construct and either left untreated or treated with BtcAMP (0.5 or 1 mM). Activity of the single copy enhancer reporter was induced 3-fold by BtcAMP treatment (Fig. 8). Activity of the double enhancer reporter was induced 11-fold by the same treatment (Fig. 8). Therefore, increased cAMP levels can increase the activity of the E3` enhancer in pre-B cells.


Figure 8: cAMP can induce activity of the E3` enhancer in pre-B cells. 1-8 pre-B cells were transfected with TKCAT constructs containing either a single or double copy of the E3` enhancer. After transfection, BtcAMP was added to a final concentration of either 0.5 or 1.0 mM.



Effect of ATF-1 and CREM on E3` Enhancer Activity

To test the influence of ATF-1 on the activity of the E3` enhancer, an ATF-1 expression plasmid was cotransfected with E3`coreTKCAT. A plasmid expressing the catalytic subunit of protein kinase A was also included (31) because ATF-1 function is activated by protein kinase A. Transfections were performed in 1-8 pre-B and S194 plasmacytoma cells. Expression of ATF-1 at various concentrations in 1-8 pre-B cells stimulated enhancer activity up to 5-fold (Fig. 9 A). In S194 plasmacytoma cells ATF-1 expression caused a 2-fold increase in enhancer activity (data not shown). However, overexpression of ATF-1 in pre-B cells did not activate the intact, normally silent E3` enhancer (data not shown). Therefore, reduced ATF-1 levels alone cannot be responsible for the lack of E3` enhancer activity at the pre-B cell stage. Interestingly, activation of the core enhancer fragment was observed at very low doses (0.5-2 ng) of ATF-1 expression plasmid. At higher concentrations, ATF-1 caused repression of the core enhancer in both cell types. Repression of enhancer activity at high ATF-1 doses suggests squelching of interacting proteins.


Figure 9: ATF-1 activates and CREM- represses activity of the E3` enhancer. Panel A, a representative CAT assay is shown of 1-8 pre-B cells cotransfected with a reporter plasmid containing the core fragment of the E3` enhancer ( 132 CORE) along with the expression plasmids encoding ATF-1 or protein kinase A ( PKA). Cells were transfected with 3 µg of reporter plasmid and 1 µg of plasmid encoding the catalytic domain of protein kinase A, 1 µg of -galactosidase expression plasmid pCH110, and various amounts of the plasmid expressing ATF-1 as indicated. The CAT values at each concentration represent averages of at least two to three independent experiments and are shown relative to the activity of the reporter plasmid. Panel B, S194 cells were cotransfected with 3 µg of the enhancer core reporter plasmid ( 132 CORE), 1 µg of internal control plasmid (pCH110; -galactosidase), and various amounts of the CREM- expression plasmid indicated below the lanes. The percent CAT activity of each construct is shown below the lanes.



Transcripts of the CREM gene can be alternatively processed to yield either transcriptional activator or repressor isoforms. We performed cotransfection experiments using expression plasmids expressing two isoforms of CREM (CREM- and CREM-). Consistent with its role as a repressor, a CREM- expression plasmid repressed enhancer activity in a dose-dependent manner (Fig. 9 B). Similar results were obtained when a plasmid expressing protein kinase A was included in the transfection. No repression of TKCAT alone was observed. Expression of the activator isoform CREM- in pre-B cells had little effect on the intact, normally silent E3` enhancer but slightly induced core enhancer activity in plasmacytoma cells (data not shown). Therefore, the repressor isoforms of CREM can repress enhancer activity, but activation of the E3` enhancer at late stages of B cell development is not controlled solely by the CREM- isoform. The above results indicate that ATF-1 and CREM proteins can influence the activity of the E3` enhancer.

ATF-1 and CREM Are Differentially Expressed in B Cells

Poly(A)RNAs isolated from cell lines representing pre-B cells (3-1 and 1-8) or plasma cells (S194 and MPC11) were subjected to Northern blot analyses with ATF-1 or CREM DNA probes. ATF-1 was found to be expressed in all cell lines with 5-6-fold greater expression in plasma cells (Fig. 10 A). Thus, ATF-1 expression appears to increase during B cell development. The same blot was stripped and rehybridized with CREM cDNA sequences. These studies showed that the CREM gene was also expressed in all cell lines studied but yielded a diffuse pattern of hybridization, presumably because of the complex processing of CREM transcripts (Fig. 10 B).


Figure 10: ATF-1 and CREM expression in B cells. Poly(A)RNAs isolated from 3-1, 1-8, MPC11, or S194 cells were subjected to Northern blot analyses and probed with ATF-1 sequences ( panel A). The same blot was stripped and rehybridized with CREM sequences ( panel B). Panel C, a diagram of the various CREM isoforms and reverse transcriptase PCR primers. In.1 and In.2 represent glutamine-rich domains, P-BOX represents a phosphorylation domain, and DNA binding domains are represented as DBDI and DBDII.



CREM transcripts can be alternatively processed to yield a variety of products (see Fig. 10C). To study CREM expression further, reverse transcriptase PCR assays were performed with RNA isolated from MPC11 plasmacytoma cells using primers A and B (see ``Materials and Methods''). The reverse transcriptase PCR products were cloned, and five independent clones were sequenced. Two of the five clones were identical to CREM-, and three represented a previously unpublished splicing variant of the CREM gene. These three clones were identical to CREM- except that they also contained a deletion of the sequences indicative of CREM- (see Fig. 10 C). The organization of this clone (CREM-/) is shown in Fig. 10C. Definitive identification of all CREM isoforms in the B cell lineage will require additional studies.

The E3`-CRE Functions Synergistically with the PU.1/NF-EM5 Binding Sites

Previously, we demonstrated that an oligonucleotide containing the E3`-CRE was inactive as an enhancer when assayed as a multimer in transient expression assays (1) . To determine whether DNA sequences adjacent to the E3`-CRE could functionally synergize in transcription, we linked several core DNA segments to the E3`-CRE and measured enhancer activities after transfection into S194 plasmacytoma cells. First, an additional 15 bp of 3`-flanking sequence that encompasses the wild type linker scan D sequences (construct E3`-CRE15) was linked to the E3`-CRE. No activity was observed when this construct was tested in S194 plasmacytoma cells (Fig. 11). We next tested whether the E3`-CRE sequence could function synergistically with either the PU.1/NF-EM5 or the E2A binding sites.


Figure 11: The E3`-CRE sequence functions synergistically with the PU.1/NF-EM5 binding sites in the 3` enhancer. S194 cells were transfected with 4 µg of the reporter plasmids indicated above each lane. Transfection efficiencies were normalized by the level of -galactosidase produced by the cotransfected plasmid pCH110. The activity of the reporter construct containing the PU.1 binding site ( PU.1TKCAT) was considered as 1, and the relative activity of the E3`-CRE+PU.1TKCAT reporter construct was determined relative to this value.



Single copies of oligonucleotides containing the PU.1/NF-EM5 or the E2A binding sites were linked adjacent to the E3`-CRE motif in the TKCAT vector. Reporter constructs containing the PU.1/NF-EM5 or E2A binding sites alone were used for comparison. Single copies of the PU.1/NF-EM5 or E2A binding sites were incapable of activating the thymidine kinase promoter (Fig. 11). Similarly, no activity was observed when the E3`-CRE was linked to the E2A binding site. However, the construct containing the E3`-CRE sequence adjacent to the PU.1/NF-EM5 binding sites resulted in 3.7-fold activation (Fig. 11). Therefore, the E3`-CRE interacts with the PU.1/NF-EM5 binding sites in the E3` enhancer to activate transcription synergistically.

ATF-1 and CREM Physically Interact with PU.1

The transcriptional synergy between theE3`-CRE and PU.1/NF-EM5 binding sites suggested that the proteins binding to these sequences might interact physically to elicit transcriptional activity. To test whether CREM and ATF-1 can physically interact with PU.1, we performed GST-fusion protein experiments. S-Labeled CREM- and ATF-1 were prepared by in vitro transcription and translation and were incubated with GST-PU.1 fusion protein bound to glutathione-agarose beads. Similar incubations were carried out with GST protein alone as a control. These experiments indicated that indeed CREM- and ATF-1 interact physically with PU.1. No interaction of CREM- or ATF-1 was observed with the GST protein alone (Fig. 12). These results are interesting because the synergistic activity observed between the E3`-CRE and PU.1/NF-EM5 binding sites might be a result of protein-protein interactions between PU.1 and ATF-1 or CREM proteins.


Figure 12: Interaction of ATF or CREM- with GST-PU.1 fusion protein. S-Labeled ATF-1 or CREM- proteins were incubated with either GST ( lanes 1 and 2) or GST-PU.1 fusion protein ( lanes 3 and 4) attached to glutathione-agarose beads. After incubation, beads were washed, suspended in SDS-sample buffer, and the bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and detected by autoradiography.




DISCUSSION

In earlier studies, we identified two functional sequences (PU.1/NF-EM5 and E2A) within the E3` enhancer core which can enhance transcription when present in multiple copies (1) . In the present study, we used a linker scan mutagenesis approach to identify additional functional enhancer sequences. This approach confirmed the importance of the PU.1/NF-EM5 and E2A binding sites for enhancer function. In addition, this linker scan approach revealed the existence of other functionally important enhancer sequences. In particular, linker scans A, B, D, and K showed reduced enhancer activities. None of these DNA sequences is capable of supporting enhancer activity when assayed as multimers (1) . Therefore, in the context of the E3` enhancer, these DNA motifs most likely bind to proteins that require interaction with other proteins to enhance transcription.

We chose to focus our efforts on the linker scan B mutation. This mutation reduced activity to near background levels when assayed in the pre-B cell line 1-8 but reduced activity to only 30% in S194 plasmacytoma cells. These results suggested a possible developmental difference in the requirement for these sequences. This functional region of the mouse E3` enhancer appears to be comparable to the DR sequence identified in the human 3` enhancer (26) . We used the mouse functional sequence as a probe to screen a cDNA expression library. This screen resulted in the isolation of the transcription factors ATF-1 and CREM. Because these factors bind to CRE elements, we named this functional sequence within the E3` enhancer the E3`-CRE. Indeed, BtcAMP was shown to increase E3` enhancer activity in pre-B cells.

Our antibody and mixing experiments (Figs. 6 and 7) indicate ATF-1 and CREM (or closely related proteins) contribute to the formation of the protein-DNA complexes observed with oligonucleotide 2. Rather than a supershift in the EMSAs, new higher mobility complexes were observed with anti-ATF-1 and anti-CREM antibodies. The CREM antibody was prepared against protein sequences that include the CREM DNA binding domain. Therefore, this antibody most likely inhibits CREM DNA binding. The ATF-1 antibodies, however, were raised against the amino-terminal 30 amino acids of ATF-1, a region distinct from the DNA binding domain. Therefore, the formation of higher mobility complexes and the lack of a supershift are somewhat surprising. One possible explanation is that ATF-1 must interact with other proteins to bind efficiently to the enhancer DNA sequences. Indeed, our mixing experiments (Fig. 7) suggest that this is the case. The anti-ATF-1 antibodies may block the ability of ATF-1 to interact with these proteins and therefore inhibit ATF-1 from binding efficiently to enhancer DNA sequences.

It is interesting that cotransfection of an ATF-1 expression plasmid increased E3` enhancer activity while a CREM expression plasmid resulted in repression. The fact that repressor as well as activator proteins bind to the E3`-CRE may help to explain why previous studies yielded conflicting results concerning the role of the cAMP signal transduction pathway in gene transcription. For instance, some reports indicated that elevation of cAMP levels within pre-B cells increased gene expression while others reported lowered expression in response to increased cAMP levels (7, 9, 16) . Our studies reported here suggest that the variety of nuclear factors that can bind to the E3`-CRE may result in complex regulatory pathways.

This issue is complicated further by the fact that the CREM gene can produce either activator or repressor proteins depending upon its mode of RNA processing, selection of transcription initiation site, or translation initiation site (see below). The initially identified isoforms, CREM-//, antagonize cAMP-induced gene expression, whereas CREM- is a transcriptional activator (29, 32, 33, 34) . In addition, a repressor form of CREM- was identified (S-CREM) which uses an internal AUG translation initiation site (35) . Recently, it was shown that the CREM gene can generate an additional product, ICER, by alternative usage of an intronic promoter in response to the adenyl cyclase signal transduction pathway (36) . ICER has been shown to be a strong repressor of cAMP-induced transcription. Therefore, differential production of the various CREM isoforms may determine whether a particular binding site functions as an activator or a repressor.

Our preliminary RNA analyses suggest that the CREM gene is expressed differentially in the B cell lineage. We performed reverse transcriptase PCR studies with RNA isolated from pre-B or plasmacytoma cells and found that pre-B and plasmacytoma cells share some CREM products but also express at least one distinct variant each.() By DNA sequence analysis, we have identified at least two CREM isoforms expressed in plasmacytoma cells (CREM- and CREM-/). One of these isoforms, CREM-/, represents a previously unpublished CREM splicing variant. A more thorough analysis will be necessary to identify all of the CREM isoforms expressed in the B cell lineage.

Our RNA studies also indicated that ATF-1 RNA levels increase during B cell development. ATF-1 was previously isolated by three independent groups based on its ability to bind to the DNA sequence CGTCA (ATF/CRE site) in a variety of viral and cellular genes (27, 28, 37) . ATF-1 is also important for Tax-mediated transactivation of the human T-cell lymphotropic virus type I long terminal repeat (38) . The activation or DNA binding properties of ATF-1 can be modulated by heterodimerization with other leucine zipper proteins (see for example, 39). Therefore, the ability of ATF-1 to augment E3` enhancer function could be influenced by the presence of other leucine zipper proteins within the cell. For instance, repressor isotypes of CREM could predominate at the pre-B cell stage when E3` enhancer activity is very weak. These repressive forms of CREM could potentially be replaced by an activator form of CREM at later stages of B cell development when enhancer activity is maximal. Alternatively, increased levels of ATF-1 may help to override inhibitory influences of the CREM gene products. The complex mode of regulation and the multiplicity of factors that bind to CRE sites (40) may explain why conflicting results have been obtained concerning the role of cAMP and gene expression (7, 9, 16) .

The functional synergism between the E3`-CRE and the PU.1/NF-EM5 binding sites is interesting. Recently, it was shown that the interleukin-6 response element of the junB promoter contains an ets binding site and a CRE-like site, which are important for efficient activation of the junB promoter by interleukin-6 (41) . These results suggest a functional interaction between leucine zipper and ets proteins. Similarly, the E3` enhancer contains a CRE-like sequence (E3`-CRE) and an ets binding site (PU.1; 42), which are located adjacent to one another. Our transfection assays indicate a functional interaction between these sequences. In addition, our glutathione S-transferase chromatography experiments (Fig. 12) indicate that PU.1 can physically interact with the proteins that bind to the E3`-CRE (ATF-1 and CREM). Therefore, the interaction of leucine zipper and ets proteins may be a common mechanism for transcriptional regulation. It will be interesting to determine the precise mechanism of PU.1 interaction with ATF-1 and CREM and how these interactions relate to the developmental control of E3` enhancer function.

  
Table:



FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1 GM42415 (to M. L. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Special fellow of the Leukemia Society of America.

To whom correspondence should be addressed: Dept. of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, Rosenthal Bldg., 3800 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-6428; Fax: 215-898-9923.

The abbreviations used are: bp, base pair(s); ATF, activating transcription factor; CRE, cAMP response element; CREM, cAMP response element modulator; TK, thymidine kinase; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay.

J. M. R. Pongubala, unpublished results.


ACKNOWLEDGEMENTS

-We thank our colleagues for critically reading the manuscript and for helpful discussions. We wish to thank G. S. McKnight for providing the expression plasmid for the catalytic domain of protein kinase A, M. Yoshida for providing the full-length Treb36 (ATF-1) expression plasmid, P. Sassone-Corsi for providing CREM- and CREM- expression plasmids and CREM antibodies, and P. Henthorn for providing the cDNA expression library. We appreciate the help of Sujatha Nagulapalli in preparing some of the plasmid constructs. We are grateful to Tom Kadesch, Narayan Avadhani, and John Pehrson for helpful comments.


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