Vol. 11, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 1040-1047 0270-7306/91/021040-08$02.00/0 Copyright © 1991, American Society for Microbiology
Functional Characterization of the Developmentally Controlled Immunoglobulin Kappa 3' Enhancer: Regulation by Id, a Repressor of Helix-Loop-Helix Transcription Factors JAGAN M. R. PONGUBALA AND MICHAEL L. ATCHISON*
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6048 Received 5 October 1990/Accepted 28 November 1990
We have functionally characterized an enhancer element (KE3') which lies 8.5 kb downstream of the immunoglobulin kappa gene. The activity of this enhancer is developmentally controlled. It is inactive at the pre-B-cell stage but active at the B-cell and plasma cell stages. This enhancer is also functional in S107 plasmacytoma cells, which lack NF-KB and therefore intron enhancer activity. The activity of the KE3' enhancer therefore provides an explanation for the transcriptional activity of endogenous kappa genes in S107 cells in the absence of intron enhancer function. We have identified a 132-bp segment of the KE3' enhancer that retains 75% of the activity of the entire enhancer observed in plasmacytoma cells. Within this 132-bp core, there are at least two functional elements, one of which binds to a B-cell-specific nuclear factor. This element contains a potential binding site for the B-cell- and macrophage-specific transcription factor PU.l. The kappa intron and KE3' enhancers were also found to be regulatable by Id, an inhibitor of helix-loop-helix transcription factors. The site of action of Id on the KE3' enhancer was mapped to a 25-bp region which contains a potential binding site for a helix-loop-helix transcription factor. A possible model for the developmental control of kappa gene transcription is discussed.
Expression of immunoglobulin kappa (IgK) genes is tissue specific. IgK transcription is controlled by a tissue-specific promoter as well as a tissue-specific enhancer (EK) which lies in the intron separating the joining from constant-region exons (10, 13, 31-34, 38). IgK genes are expressed in the B-lymphoid developmental pathway and are developmentally regulated. They are transcriptionally silent at the preB-cell stage but are transcriptionally active at the B-cell and plasma cell stages. Treatment of pre-B-cell lines with bacterial lipopolysaccaride (LPS), however, induces their transcriptional activity (29, 30, 39). This activation is due to the induction of a trans-acting nuclear factor, NF-KB, that is crucial for intron enhancer activity (la, 24, 35, 36). Latestage cells (B cell and plasma cell) constitutively express NF-KB (35), and therefore the intron enhancer is constitutively active. Plasmacytoma S107 cells, however, lack functional NF-KB yet continue to express their endogenous kappa genes (la). Transfected genes under the control of the intron enhancer are silent in this cell line, demonstrating the inactivity of the intron enhancer. These results were interpreted to indicate that kappa locus transcription is differentially regulated during B-cell development. Possible models of apparent enhancer-independent regulation included the production of a stable, heritable transcription complex, the developmental alteration of kappa locus chromatin into a transcriptionally active conformation (possibly relating to DNA methylation status), or the existence of distal regulatory elements outside of the DNA sequences used in transfection assays (la, 2, 22). Recently an additional enhancer element (KE3') was identified 8.5 kb downstream of the constant-region exon (25). This enhancer was found to be B cell specific and could *
potentially account for the transcriptional activity of the endogenous kappa genes in S107 plasmacytoma cells, which lack NF-KB. While this 3' enhancer was shown to be active in cell lines representative of the B-cell and plasma cell stages (25), its activity in pre-B-cell lines is unreported. We show here that the KE3' enhancer is active in S107 plasmacytoma cells, suggesting that it is likely to be responsible for maintaining endogenous kappa transcription in the absence of intron enhancer function. We also show that the KE3' enhancer is developmentally controlled, being inactive at the pre-B-cell stage but active at the B-cell and plasma cell stages. We have functionally characterized the 3' enhancer in plasmacytoma cells and have defined a 132-bp core that retains about 75% of the activity of the intact enhancer. We show that activity of the intact 3' enhancer as well as the 132-bp core can be inhibited by expression of Id, an inhibitor of helix-loop-helix (HLH) transcription factors. Within the 132-bp core there are at least two functional elements, one of which is inhibited by Id, while the other binds a nuclear factor that is present in B-lymphoid cell extracts but absent in 3T3 cells. MATERIALS AND METHODS Plasmid constructions. TKCAT is composed of sequences -109 to +55 of the herpesvirus thymidine kinase (TK) promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and the simian virus 40 splice and polyadenylation sequences. TKCAT was linearized at the Sall site just upstream of the TK promoter, filled in with Klenow polymerase, and ligated to a blunted 1.1-kb SaclEcoRI DNA fragment containing the KE3' enhancer to produce KE3'TKCAT or to a blunted 2-kb BanIl fragment containing the intron enhancer to produce EKTKCAT. For plasmid constructs A through E (see Fig. 4), the following DNA fragments were cloned by blunt-end ligation into the
Corresponding author. 1040
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DEVELOPMENTALLY CONTROLLED KE3' ENHANCER
HincII site of pUC18: A, a 443-bp BglII-ApaLI DNA fragment; B, a 311-bp BgIII-AvaII DNA fragment; C, a 415-bp AvaII-XbaI DNA fragment; D, a 285-bp ApaLI-XbaI DNA fragment; and E, a 132-bp AvaII-ApaLI DNA fragment. These DNA fragments were excised from pUC18 DNA sequences by HindIII-BamHI digestion, purified by polyacrylamide gel electrophoresis, and ligated into the HindIIIBamHI sites upstream of the TK promoter in TKCAT. For plasmids F through K, the 729-bp BglII-XbaI DNA fragment containing the 3' enhancer was digested with HaeIII and DdeI. The resulting DNA fragments were purified by polyacrylamide gel electrophoresis, blunted with Klenow polymerase, and ligated into the HinclI site of pUC18. The resulting subclones were transferred into HindIII-BamHIcut TKCAT as described above. Clones F through J contain two head-to-tail copies of the inserted DNA fragment, while clone K contains five copies. To produce clones L and M, the 125-bp DdeI fragment which constitutes the DNA sequences in clone H was cut into two portions with AluI, and the resulting DNA fragments were inserted into the HincII site of pUC18 and transferred to TKCAT as described above. Similarly, to produce plasmids N and 0, the 114-bp DdeI-HaeIII DNA fragment which constitutes the DNA sequences in clone I was cut into two fragments by BstNI digestion, and each fragment was cloned as described above. Plasmids M and N contained two copies of the enhancer DNA sequences, while plasmids L and 0 contained three copies. To produce plasmids 1 through 9 (see Fig. 5), oligonucleotides were prepared with BamHI and BgII termini. These oligonucleotides were inserted into the BamHI and BglII sites of plasmid LBKA44 (kindly supplied by D. Ruezinsky, University of Pennsylvania). Each plasmid was cut either with BamHI and BglI (which cuts in the body of the plasmid) or with BglII and BglI. The BamHI-BglI and Bg[II-Bgll DNA fragments were purified and ligated to produce a plasmid with a duplicated copy of the oligonucleotide sequence. The procedure was repeated to generate plasmids with four copies. Each clone was digested with BamHI and BglII, and the DNA fragments containing the multimerized oligonucleotides were purified by polyacrylamide gel electrophoresis and subcloned into the BamHI site upstream of the TK promoter in TKCAT to generate constructs 1 through 9 (see Fig. 5). The DNA content of all constructs was verified by DNA sequence analysis. Cell culture and transfections. S194 cells were grown in Dulbecco modified Eagle medium supplemented with 10% horse serum (GIBCO). 3-1, 1-8, 38C, and S107 cells were grown in RPMI supplemented with 10% fetal calf serum (GIBCO) and 50 ,uM 2-mercaptoethanol. Transfections were performed by the DEAE-dextran (Pharmacia) procedure according to Grosschedl and Baltimore (16) except that treatment with chloroquine diphosphate was omitted. Transfection efficiencies were monitored by cotransfection with the ,-galactosidase expression plasmid pCH110 (17) or by measurement of the transfected plasmid DNA in the Hirt supernatant fraction (21). Cells were harvested after 44 h (S194, S107, and 38C cells) or 26 h (3-1 and 1-8 cells), and CAT extracts were prepared. CAT assays and thin-layer chromatography were performed according to Gorman et al. (15). CAT activities were quantitated by liquid scintillation counting of the reaction products separated by thin-layer chromatography. For Id cotransfection studies, all samples contained 0.5 ,ug of the reporter plasmid and 1 ,ug of the ,-galactosidase plasmid pCH110. Each sample then contained 4 ,ug of either a plasmid expressing the Id cDNA, a
1041
3'enhancer
A intron
(E3') Sect EcoRI
enhnw (Ec)
I I 1.0kb
iI
B
swETTKCAT
ft,
%
J.
%
PrK
%I.J- 11%
CAT
%.Vl%g
TKCAT /.
%
EXTKCAT Pt FIG. 1. (A) Map of the downstream portion of the kappa locus. The filled-in box indicates the position of the kappa constant region exon (CK), and circles show the positions of the intron (EK) and 3'
(KE3') enhancers. Positions of the Sacl and EcoRI sites flanking the KE3' enhancer are indicated. (B) Map of DNA constructs. Symbols: C, DNA sequences -109 to +55 of the herpesvirus TK promoter; _, bacterial CAT gene; =I, simian virus 40 splice and polyadenylation region; , pUC8 DNA sequences. The positions and orientations of the KE3' and intron enhancer DNA fragments are indicated.
plasmid with the Id sequences in reverse orientation (3), or pUC18 DNA. Electrophoretic mobility shift assays. Assays were performed with 32P-end-labeled oligonucleotides according to Singh et al. (37). Nuclear extracts were prepared from S194, 3-1, and 3T3 cells according to Dignam et al. (7). Unlabeled competitors were added in the amounts indicated on the figures. RESULTS We sought to determine the developmental activity of the KE3' enhancer and whether it is active in S107 cells, which lack NF-KB. The KE3' enhancer lies 8.5 kb downstream of CK and is flanked by SacI and EcoRI sites (Fig. 1A). This 1.1-kb SacI-EcoRI DNA fragment was inserted into vector TKCAT, which contains DNA sequences -109 to +55 of the herpesvirus TK promoter linked to the bacterial CAT gene to produce plasmid KE3'TKCAT (Fig. 1B). Similarly, a 2-kb BanII DNA fragment containing the kappa intron enhancer was inserted into TKCAT to produce EKTKCAT (Fig. 1B). The transcriptional activities of these plasmids were determined after transfection into cell lines of defined developmental stage. The cE3' enhancer is active in S107 plasmacytoma cells. The DNA constructs shown in Fig. 1B were individually transfected into S107 plasmacytoma cells. A CAT construct controlled by the Rous sarcoma virus long terminal repeat (RSVCAT; 14) was included as a positive control. As expected, the enhancerless construct (TKCAT) was inactive (Fig. 2, lane 1), as was the construct containing the intron enhancer (EKTKCAT; Fig. 2, lane 2) as a result of the absence of NF-KB in these cells. In contrast, the DNA construct containing the KE3' enhancer (KE3'TKCAT) caused a 35-fold stimulation in expression (Fig. 2, lane 3), which was 4-fold stronger than that of RSVCAT (Fig. 2, lane 4). These results indicate that the KE3' enhancer is indeed active in S107 plasmacytoma cells and therefore does not require NF-KB for activity. Meyer and Neuberger (25) demonstrated that the KE3' enhancer is active at the B-cell
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MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 1040-1047 0270-7306/91/021040-08$02.00/0 Copyright © 1991, American Society for Microbiology
Functional Characterization of the Developmentally Controlled Immunoglobulin Kappa 3' Enhancer: Regulation by Id, a Repressor of Helix-Loop-Helix Transcription Factors JAGAN M. R. PONGUBALA AND MICHAEL L. ATCHISON*
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6048 Received 5 October 1990/Accepted 28 November 1990
We have functionally characterized an enhancer element (KE3') which lies 8.5 kb downstream of the immunoglobulin kappa gene. The activity of this enhancer is developmentally controlled. It is inactive at the pre-B-cell stage but active at the B-cell and plasma cell stages. This enhancer is also functional in S107 plasmacytoma cells, which lack NF-KB and therefore intron enhancer activity. The activity of the KE3' enhancer therefore provides an explanation for the transcriptional activity of endogenous kappa genes in S107 cells in the absence of intron enhancer function. We have identified a 132-bp segment of the KE3' enhancer that retains 75% of the activity of the entire enhancer observed in plasmacytoma cells. Within this 132-bp core, there are at least two functional elements, one of which binds to a B-cell-specific nuclear factor. This element contains a potential binding site for the B-cell- and macrophage-specific transcription factor PU.l. The kappa intron and KE3' enhancers were also found to be regulatable by Id, an inhibitor of helix-loop-helix transcription factors. The site of action of Id on the KE3' enhancer was mapped to a 25-bp region which contains a potential binding site for a helix-loop-helix transcription factor. A possible model for the developmental control of kappa gene transcription is discussed.
Expression of immunoglobulin kappa (IgK) genes is tissue specific. IgK transcription is controlled by a tissue-specific promoter as well as a tissue-specific enhancer (EK) which lies in the intron separating the joining from constant-region exons (10, 13, 31-34, 38). IgK genes are expressed in the B-lymphoid developmental pathway and are developmentally regulated. They are transcriptionally silent at the preB-cell stage but are transcriptionally active at the B-cell and plasma cell stages. Treatment of pre-B-cell lines with bacterial lipopolysaccaride (LPS), however, induces their transcriptional activity (29, 30, 39). This activation is due to the induction of a trans-acting nuclear factor, NF-KB, that is crucial for intron enhancer activity (la, 24, 35, 36). Latestage cells (B cell and plasma cell) constitutively express NF-KB (35), and therefore the intron enhancer is constitutively active. Plasmacytoma S107 cells, however, lack functional NF-KB yet continue to express their endogenous kappa genes (la). Transfected genes under the control of the intron enhancer are silent in this cell line, demonstrating the inactivity of the intron enhancer. These results were interpreted to indicate that kappa locus transcription is differentially regulated during B-cell development. Possible models of apparent enhancer-independent regulation included the production of a stable, heritable transcription complex, the developmental alteration of kappa locus chromatin into a transcriptionally active conformation (possibly relating to DNA methylation status), or the existence of distal regulatory elements outside of the DNA sequences used in transfection assays (la, 2, 22). Recently an additional enhancer element (KE3') was identified 8.5 kb downstream of the constant-region exon (25). This enhancer was found to be B cell specific and could *
potentially account for the transcriptional activity of the endogenous kappa genes in S107 plasmacytoma cells, which lack NF-KB. While this 3' enhancer was shown to be active in cell lines representative of the B-cell and plasma cell stages (25), its activity in pre-B-cell lines is unreported. We show here that the KE3' enhancer is active in S107 plasmacytoma cells, suggesting that it is likely to be responsible for maintaining endogenous kappa transcription in the absence of intron enhancer function. We also show that the KE3' enhancer is developmentally controlled, being inactive at the pre-B-cell stage but active at the B-cell and plasma cell stages. We have functionally characterized the 3' enhancer in plasmacytoma cells and have defined a 132-bp core that retains about 75% of the activity of the intact enhancer. We show that activity of the intact 3' enhancer as well as the 132-bp core can be inhibited by expression of Id, an inhibitor of helix-loop-helix (HLH) transcription factors. Within the 132-bp core there are at least two functional elements, one of which is inhibited by Id, while the other binds a nuclear factor that is present in B-lymphoid cell extracts but absent in 3T3 cells. MATERIALS AND METHODS Plasmid constructions. TKCAT is composed of sequences -109 to +55 of the herpesvirus thymidine kinase (TK) promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and the simian virus 40 splice and polyadenylation sequences. TKCAT was linearized at the Sall site just upstream of the TK promoter, filled in with Klenow polymerase, and ligated to a blunted 1.1-kb SaclEcoRI DNA fragment containing the KE3' enhancer to produce KE3'TKCAT or to a blunted 2-kb BanIl fragment containing the intron enhancer to produce EKTKCAT. For plasmid constructs A through E (see Fig. 4), the following DNA fragments were cloned by blunt-end ligation into the
Corresponding author. 1040
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1991
DEVELOPMENTALLY CONTROLLED KE3' ENHANCER
HincII site of pUC18: A, a 443-bp BglII-ApaLI DNA fragment; B, a 311-bp BgIII-AvaII DNA fragment; C, a 415-bp AvaII-XbaI DNA fragment; D, a 285-bp ApaLI-XbaI DNA fragment; and E, a 132-bp AvaII-ApaLI DNA fragment. These DNA fragments were excised from pUC18 DNA sequences by HindIII-BamHI digestion, purified by polyacrylamide gel electrophoresis, and ligated into the HindIIIBamHI sites upstream of the TK promoter in TKCAT. For plasmids F through K, the 729-bp BglII-XbaI DNA fragment containing the 3' enhancer was digested with HaeIII and DdeI. The resulting DNA fragments were purified by polyacrylamide gel electrophoresis, blunted with Klenow polymerase, and ligated into the HinclI site of pUC18. The resulting subclones were transferred into HindIII-BamHIcut TKCAT as described above. Clones F through J contain two head-to-tail copies of the inserted DNA fragment, while clone K contains five copies. To produce clones L and M, the 125-bp DdeI fragment which constitutes the DNA sequences in clone H was cut into two portions with AluI, and the resulting DNA fragments were inserted into the HincII site of pUC18 and transferred to TKCAT as described above. Similarly, to produce plasmids N and 0, the 114-bp DdeI-HaeIII DNA fragment which constitutes the DNA sequences in clone I was cut into two fragments by BstNI digestion, and each fragment was cloned as described above. Plasmids M and N contained two copies of the enhancer DNA sequences, while plasmids L and 0 contained three copies. To produce plasmids 1 through 9 (see Fig. 5), oligonucleotides were prepared with BamHI and BgII termini. These oligonucleotides were inserted into the BamHI and BglII sites of plasmid LBKA44 (kindly supplied by D. Ruezinsky, University of Pennsylvania). Each plasmid was cut either with BamHI and BglI (which cuts in the body of the plasmid) or with BglII and BglI. The BamHI-BglI and Bg[II-Bgll DNA fragments were purified and ligated to produce a plasmid with a duplicated copy of the oligonucleotide sequence. The procedure was repeated to generate plasmids with four copies. Each clone was digested with BamHI and BglII, and the DNA fragments containing the multimerized oligonucleotides were purified by polyacrylamide gel electrophoresis and subcloned into the BamHI site upstream of the TK promoter in TKCAT to generate constructs 1 through 9 (see Fig. 5). The DNA content of all constructs was verified by DNA sequence analysis. Cell culture and transfections. S194 cells were grown in Dulbecco modified Eagle medium supplemented with 10% horse serum (GIBCO). 3-1, 1-8, 38C, and S107 cells were grown in RPMI supplemented with 10% fetal calf serum (GIBCO) and 50 ,uM 2-mercaptoethanol. Transfections were performed by the DEAE-dextran (Pharmacia) procedure according to Grosschedl and Baltimore (16) except that treatment with chloroquine diphosphate was omitted. Transfection efficiencies were monitored by cotransfection with the ,-galactosidase expression plasmid pCH110 (17) or by measurement of the transfected plasmid DNA in the Hirt supernatant fraction (21). Cells were harvested after 44 h (S194, S107, and 38C cells) or 26 h (3-1 and 1-8 cells), and CAT extracts were prepared. CAT assays and thin-layer chromatography were performed according to Gorman et al. (15). CAT activities were quantitated by liquid scintillation counting of the reaction products separated by thin-layer chromatography. For Id cotransfection studies, all samples contained 0.5 ,ug of the reporter plasmid and 1 ,ug of the ,-galactosidase plasmid pCH110. Each sample then contained 4 ,ug of either a plasmid expressing the Id cDNA, a
1041
3'enhancer
A intron
(E3') Sect EcoRI
enhnw (Ec)
I I 1.0kb
iI
B
swETTKCAT
ft,
%
J.
%
PrK
%I.J- 11%
CAT
%.Vl%g
TKCAT /.
%
EXTKCAT Pt FIG. 1. (A) Map of the downstream portion of the kappa locus. The filled-in box indicates the position of the kappa constant region exon (CK), and circles show the positions of the intron (EK) and 3'
(KE3') enhancers. Positions of the Sacl and EcoRI sites flanking the KE3' enhancer are indicated. (B) Map of DNA constructs. Symbols: C, DNA sequences -109 to +55 of the herpesvirus TK promoter; _, bacterial CAT gene; =I, simian virus 40 splice and polyadenylation region; , pUC8 DNA sequences. The positions and orientations of the KE3' and intron enhancer DNA fragments are indicated.
plasmid with the Id sequences in reverse orientation (3), or pUC18 DNA. Electrophoretic mobility shift assays. Assays were performed with 32P-end-labeled oligonucleotides according to Singh et al. (37). Nuclear extracts were prepared from S194, 3-1, and 3T3 cells according to Dignam et al. (7). Unlabeled competitors were added in the amounts indicated on the figures. RESULTS We sought to determine the developmental activity of the KE3' enhancer and whether it is active in S107 cells, which lack NF-KB. The KE3' enhancer lies 8.5 kb downstream of CK and is flanked by SacI and EcoRI sites (Fig. 1A). This 1.1-kb SacI-EcoRI DNA fragment was inserted into vector TKCAT, which contains DNA sequences -109 to +55 of the herpesvirus TK promoter linked to the bacterial CAT gene to produce plasmid KE3'TKCAT (Fig. 1B). Similarly, a 2-kb BanII DNA fragment containing the kappa intron enhancer was inserted into TKCAT to produce EKTKCAT (Fig. 1B). The transcriptional activities of these plasmids were determined after transfection into cell lines of defined developmental stage. The cE3' enhancer is active in S107 plasmacytoma cells. The DNA constructs shown in Fig. 1B were individually transfected into S107 plasmacytoma cells. A CAT construct controlled by the Rous sarcoma virus long terminal repeat (RSVCAT; 14) was included as a positive control. As expected, the enhancerless construct (TKCAT) was inactive (Fig. 2, lane 1), as was the construct containing the intron enhancer (EKTKCAT; Fig. 2, lane 2) as a result of the absence of NF-KB in these cells. In contrast, the DNA construct containing the KE3' enhancer (KE3'TKCAT) caused a 35-fold stimulation in expression (Fig. 2, lane 3), which was 4-fold stronger than that of RSVCAT (Fig. 2, lane 4). These results indicate that the KE3' enhancer is indeed active in S107 plasmacytoma cells and therefore does not require NF-KB for activity. Meyer and Neuberger (25) demonstrated that the KE3' enhancer is active at the B-cell
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