Proc. Nati. Acad. Sci. USA Vol. 88, pp. 9804-9808, November 1991 Biochemistry
Isolation of a candidate repressor/activator, NF-E1 (YY-1, 6), that binds to the immunoglobulin Kc 3' enhancer and the immunoglobulin heavy-chain IAE1 site (transcription/developmental control/negative regulation)
KYOUNGSOOK PARK AND MICHAEL L. ATCHISON Department of Animal Biology, Biomedical Graduate Studies, Molecular Biology Program, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104
Communicated by Robert P. Perry, August 7, 1991
ABSTRACT We have determined that the developmental control of immunoglobulin K 3' enhancer (cE3') activity is the result ofthe combined influence of positive- and negative-acting elements. We show that a central core in the dcE3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position- and orientationindependent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the KE3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer ,uE1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse 8 protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed.
132-bp core we have identified a segment that represses activity of the enhancer at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to a DNA sequence within the negativeacting segment. This factor also binds to the juE1 site of the heavy-chain enhancer. The sequence of NF-E1 reveals features in common with both transcriptional activators as well as transcriptional repressors.* Cotransfection with this cDNA results in repression of basal promoter activity.
MATERIALS AND METHODS Plasmid Constructions. Construct A contains a Bgl II/Xba I DNA fragment (residues 80-808) of the KE3' enhancer upstream of the herpesvirus thymidine kinase (TK) promoter and the chloramphenicol acetyltransferase (CAT) sequences. Plasmids B-G were prepared either by BAL-31 deletion or by PCR amplification of appropriate portions of the KE3' enhancer. Plasmid J was prepared by a PCR-based linker scan procedure (12) such that the NF-E1 sequence was changed from CCTCCATC to AGAATTCA. Plasmids H and I were described previously (6). The GAL4-E1 and GAL4-rE1 fusion constructs contain the insert from clone 324 in either orientation at the EcoRI site of plasmid pSG424 (13). SV-E1 and SV-rEl contain the clone 131 cDNA in either orientation driven by the simian virus 40 promoter and enhancer. Cell Culture, Transfections, and CAT Assays. 1-8 cells were grown and transfected by the DEAE-dextran procedure as described (6). 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10%6 heat-inactivated horse serum (GIBCO) and were transfected by the calcium phosphate coprecipitation method (14). 3T3 transfections contained 4 jug of reporter plasmid, 15 jg of effector plasmid, and 1 Rug of the f3-galactosidase expression plasmid pCH110 (15). CAT assays were performed as described by Gorman et al. (16). Gel Mobility Shift and Methylation Interference Assays. Electrophoretic mobility shift assays were performed as described by Singh et al. (17). For methylation interference assays, a 75-bp Hae III/BstNI fragment containing the NF-E1 binding site was subcloned into the HincIl site of pUC18. This plasmid was cut with BamHI, dephosphorylated with calf intestine phosphatase, and then labeled with [y32P]ATP by polynucleotide kinase. Partial methylation, binding reactions with Ag8 nuclear extract, and electrophoresis were performed as described (18). Screening of the cDNA Expression Library. A human B-cellderived Agtll cDNA library (human B-cell leukemia RPM
Immunoglobulin K gene expression is tissue specific and is developmentally regulated during B-cell development at the transcriptional level. Immunoglobulin K transcription is governed by two enhancers: one (EK) lies within the intron separating the joining and constant regions, and the other (KE3') lies downstream of the constant region (1-3). Both enhancers are tissue specific and both are developmentally controlled (3-6). That is, both enhancers are normally silent at the pre-B-cell stage but are active at the B-cell and plasma cell stages. The activity of the intron enhancer is controlled by the presence of the active form of NF-KB (7-10). The 3' enhancer, however, does not require NF-KB for its activity (6, 11). Therefore, the mechanism of its silence at the pre-B-cell stage is unclear. Silence of the KE3' enhancer at the pre-B-cell stage could be due to either the lack of positive-acting factors, the presence of negative-acting factors, or both. Previously, we functionally characterized the KE3' enhancer and identified a 132-base-pair (bp) core that retains 75% of the activity of the intact 1-kilobase (kb) enhancer in plasmacytoma cells (6). Sequences flanking this 132-bp core contribute very little to enhancer activity at this stage of B-cell development. It is unknown whether sequences within this core are capable of being active at the pre-B-cell stage of development or whether there are negative-acting elements in the KE3' enhancer. We report here that the KE3' enhancer 132-bp core is indeed active at the pre-B-cell stage. Downstream of the
Abbreviations: TK, thymidine kinase; CAT, chloramphenicol acetyltransferase. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M76541).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9804
Biochemistry: Park and Atchison
Proc. Natl. Acad. Sci. USA 88 (1991)
4265; Clontech) was screened with a labeled multimerized DNA binding site oligonucleotide according to the method of Vinson et al. (19). The oligonucleotide used for screening is GATCCTACCCCACCTCCATCTTGTTTGATA GATGGGGTGGAGGTAGAACAAACTATCTAG
DNA Sequence Analysis. DNA sequences were determined by the dideoxynucleotide chain-termination method on double-stranded DNA templates using the United States Biochemical sequencing kit. In Vitro Transcription and Translation. Clone 131 in pBluescript was linearized with Xba I and then transcribed with T7 polymerase using an mRNA capping kit according to the manufacturer's specifications (Stratagene). The capped mRNA was subjected to in vitro translation with a rabbit reticulocyte lysate (Promega).
RESULTS The E3' Core Is Active in pre-B Ceils and Is Flanked by Negative-Acting Elements. We set out to determine whether the 132-bp KE3' core is active at the pre-B-cell stage. The 132-bp core segment was linked to the herpesvirus TK promoter driving expression of the CAT gene. The activity of this construct was compared to that of the intact enhancer contained on a 728-bp DNA fragment (containing 311 bp 5' and 285 bp 3' of the core) after transfection into 1-8 pre-B cells. The 132-bp core segment was -10-fold more active than the 728-bp enhancer fragment in pre-B cells (Fig. 1A, compare constructs A and I). These results suggest the presence of negative regulatory elements within the 728-bp enhancer fragment that are deleted in the 132-bp fragment. To search for these negative elements, various deletion mutants were prepared (Fig. 1A). Deletion of sequences between positions 808 and 658 did not increase activity of the enhancer
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FIG. 1. Identification of negative-acting segments in the KE3' enhancer. (A) DNA sequences present in each construct are shown on the left and are represented by open boxes. The relative activity of each construct in 1-8 pre-B cells is indicated on the right. Standard deviations were calculated from three to five transfection experiments. In construct J, x x indicates the position of a linker scan mutation in the NF-E1 binding site. (B) Position and orientation of sequences 523-808 inserted into construct H are indicated by arrows. CAT activities relative to construct H are shown.
9805
above that of the intact enhancer (Fig. 1A, compare constructs A, B, and C). However, progressive deletions to positions 615, 558, 536, and 523 (constructs D, F, G, and H, respectively) resulted in stepwise increases in CAT activity. Similar results were obtained with the pre-B-cell line 3-1 (data not shown). These results suggest the existence of multiple negative control elements that flank the 3' side of the 132-bp core of the KE3' enhancer. To ensure that the increased enhancer activity was not due to position effects caused by the various deletions and to determine whether the negative-acting segment was position or orientation independent, constructs K-N were prepared (Fig. 1B). Construct H was modified to contain sequences 523-808 on either the 3' side (K and L) or the 5' side (M and N) of the enhancer. All four constructs repressed enhancer activity, indicating that the negative-acting sequences are position and orientation independent (Fig. 1B). Repression ranged from 9- to 3.4-fold (11-29% of construct H). Identification of a Nuclear Factor That Binds to the NegativeActing Region of the ,cE3' Enhancer. To identify nuclear factors that bind to the negative-acting region of the KE3' enhancer DNA fragments were used as probes in electrophoretic mobility shift assays. A 75-bp Hae III/BstNI DNA fragment (residues 582-657) yielded a single bound complex with nuclear extracts prepared from cell lines representative of the pre-B-cell, B-cell, and plasma cell stages (data not shown). Unlabeled DNA fragments from the KE3' and the heavy-chain enhancers abolished protein-DNA interaction, but a DNA fragment from the K intron enhancer had no effect (data not shown). Oligonucleotides containing known immunoglobulin enhancer motifs were also used as competitors. An oligonucleotide containing the AE1 binding site abolished the DNA-nuclear factor interaction (Fig. 2 Upper), but not oligonucleotides containing the /ME2, IE3, ,uE4, puE5, NFKB, or octamer binding sites (data not shown). Thus, the nuclear factor that binds to the KE3' probe appears to be identical to the factor that binds to the immunoglobulin heavy-chain ME1 site. We therefore named this nuclear factor NF-E1. Dimethyl sulfate methylation interference assays indicated that methylation of G residues 623, 624, 626, 627, and 630 on the bottom DNA strand of the KE3' enhancer interfered with NF-E1-DNA interaction (Fig. 2 Left). These residues in the KE3' enhancer overlap with a region of 8 bp of identity with the heavy-chain ,uE1 site and closely correspond to sites identified by in vivo and in vitro ,E1 dimethyl sulfate footprinting (refs. 20 and 21; summarized in Fig. 2). Isolation and Characterization of cDNA Clones Encoding NF-El. A human B-cell cDNA library in Agtll was screened with an oligonucleotide containing the KE3' NF-E1 binding sequence by the method of Vinson et al. (19). Of 250,000 phage plaques screened, we obtained 3 positive clones designated 131, 324, and 1021. The largest cDNA clone (clone 324) was sequenced in its entirety and contains 2084 bp. Clone 131, although smaller than clone 324, extended the sequence an additional 92 bp in the 5' direction. The composite NF-E1 sequence spans 2176 bp and, beginning with the first ATG triplet at position 75, encodes an open reading frame of 414 amino acids (Fig. 3). The first methionine codon conforms well to the Kozak (22) consensus sequence. The termination codon is followed by 860 bp of 3' untranslated sequence. A polyadenylylation signal lies 4 bp upstream of the 3' end of the cDNA sequence. A second potential polyadenylylation signal lies 58 bp downstream of the translational termination codon. Analysis of the predicted amino acid sequence revealed a rather unusual structure. First, in the C-terminal portion are four zinc fingers of the Cis-Cis-His-His type. Second, the N-terminal portion of the protein is highly acidic with 21 of the first 53 (40%) amino acids being acidic. This region
9806
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Park and Atchison
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includes a portion with 11 consecutive acid residues between amino acids 43 and 53. The segment between residues 16 and 35 contains sequences consistent with the ability to form a negatively charged amphipathic a-helix. This highly acidic N terminus is reminiscent of transcriptional activation domains found in other transcriptional regulators (23, 24). Third, the segment between residues 70 and 80 is unusual in that it consists of 11 consecutive histidines. The transcriptional repressor Tst-1 (SCIP) also contains regions rich in histidine as well as a stretch of 6 consecutive histidine residues (25, 26). Fourth, the region between amino acids 154 and 198 is very rich in alanine and glycine residues (26 of 45; 58%). This region also includes a stretch of 4 consecutive serines between residues 164 and 167. The repressor Tst-1 (SCIP) also contains extensive regions rich in alanine and glycine (45%). In addition, the putative repressor domain in the transcniptional repressor Kruppel is rich in alanine residues (27). A search of the National Biomedical Research FoundationProtein Identification Resource (release 26) data base revealed homologies to numerous transcription factor IIIAtype finger proteins. Particularly striking was a 76% homology in the zinc finger region to the finger protein REX-i (28). No data base homologies to other segments of the NF-E1 sequence were observed.
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FIG. 3. DNA and predicted amino acid sequence of NF-E1. Amino acid and nucleotide coordinates are indicated on the right. Histidine residues are circled. Alanine and glycine residues are underlined. The two boxed regions denote the locations of the 11 consecutive acid residues near the N terminus and the zinc finger region at the C terminus. Asterisk shows the location of the termination codon. The 5' and 3' untranslated sequences are shown in italics. The two underlined sequences in the 3' untranslated region indicate potential polyadenylylation signals.
The sequences of clones 131 and 324 were compared over -800 bp. Both sequences were identical except at two locations. At nucleotide position 268, a G to A transition results in an arginine to histidine change in clone 324. In addition, a CCA codon is deleted in clone 324 between nucleotide positions 271 and 273, resulting in the loss of a single histidine residue. These differences presumably reflect polymorphisms of the same gene. DNA Binding Characterization of the Cloned and Endogenous NF-El Proteins. Clone 131 was transcribed and translated in vitro and the synthesized protein was assayed for binding to the KE3' NF-E1 DNA probe. The in vitro synthesized protein bound to the KE3' NF-E1 probe and migrated with a mobility identical to the endogenous protein (Fig. 4A, lanes 2 and 3). The protein-DNA complex was abolished by competition with unlabeled oligonucleotides containing the KE3' NF-E1 or the immunoglobulin heavy-chain ,E1 binding sites, but not with one containing a mutant NF-E1 sequence (lanes 4-6). The zinc chelator 1,10-phenanthroline (29) abolished DNA binding by the in vitro synthesized and the endogenous proteins (Fig. 4A, lane 7; Fig. 4B, lanes 1 and 2).
Biochemistry: Park and Atchison
Proc. Natl. Acad. Sci. USA 88 (1991)
in the opposite orientation (GAL4-rEl) or the GAL4 1-147 sequences alone (GAL4 1-147) had no effect on expression (lanes 2 and 4). The above repression could be due to NF-E1 directly repressing promoter activity. Alternatively, NF-E1 could be titrating factors necessary for promoter activity (squelching). To distinguish between these possibilities, we cotransfected the various effector constructs with a reporter plasmid that lacked the GAL4 DNA binding site (TKCAT). TKCAT expression was unaffected by cotransfection with the GAL4-El construct, indicating that DNA binding is necessary for the repressive activity of NF-E1 (Fig. 5, lanes 5-8). To ensure that the repression observed above was not due to an unusual feature of the GAL4-El chimera, we expressed the native NF-E1 cDNA without GAL4 sequences. The NF-E1 expression plasmid (SV-E1) repressed activity of a TKCAT reporter containing the NF-E1 binding site, (E1)4TKCAT, while an expression plasmid with the NF-E1 sequence in the opposite orientation (SV-rE1) had no effect (lanes 9-11). To assess the functional role of the NF-El binding site in the context of the intact KE3' enhancer, we prepared a linker scan mutation of the NF-El binding site. This construct gave a small, but reproducible, 1.3-fold activation of the intact KE3' enhancer in pre-B cells (Fig. 1A, construct J). This low level of activation was not unexpected given our evidence that the KE3' enhancer contains multiple negative elements (Fig. 1A). Our results are consistent with the NF-E1 site playing a negative role in KE3' enhancer activity rather than a positive role.
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Isolation of a candidate repressor/activator, NF-E1 (YY-1, 6), that binds to the immunoglobulin Kc 3' enhancer and the immunoglobulin heavy-chain IAE1 site (transcription/developmental control/negative regulation)
KYOUNGSOOK PARK AND MICHAEL L. ATCHISON Department of Animal Biology, Biomedical Graduate Studies, Molecular Biology Program, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104
Communicated by Robert P. Perry, August 7, 1991
ABSTRACT We have determined that the developmental control of immunoglobulin K 3' enhancer (cE3') activity is the result ofthe combined influence of positive- and negative-acting elements. We show that a central core in the dcE3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position- and orientationindependent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the KE3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer ,uE1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse 8 protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed.
132-bp core we have identified a segment that represses activity of the enhancer at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to a DNA sequence within the negativeacting segment. This factor also binds to the juE1 site of the heavy-chain enhancer. The sequence of NF-E1 reveals features in common with both transcriptional activators as well as transcriptional repressors.* Cotransfection with this cDNA results in repression of basal promoter activity.
MATERIALS AND METHODS Plasmid Constructions. Construct A contains a Bgl II/Xba I DNA fragment (residues 80-808) of the KE3' enhancer upstream of the herpesvirus thymidine kinase (TK) promoter and the chloramphenicol acetyltransferase (CAT) sequences. Plasmids B-G were prepared either by BAL-31 deletion or by PCR amplification of appropriate portions of the KE3' enhancer. Plasmid J was prepared by a PCR-based linker scan procedure (12) such that the NF-E1 sequence was changed from CCTCCATC to AGAATTCA. Plasmids H and I were described previously (6). The GAL4-E1 and GAL4-rE1 fusion constructs contain the insert from clone 324 in either orientation at the EcoRI site of plasmid pSG424 (13). SV-E1 and SV-rEl contain the clone 131 cDNA in either orientation driven by the simian virus 40 promoter and enhancer. Cell Culture, Transfections, and CAT Assays. 1-8 cells were grown and transfected by the DEAE-dextran procedure as described (6). 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10%6 heat-inactivated horse serum (GIBCO) and were transfected by the calcium phosphate coprecipitation method (14). 3T3 transfections contained 4 jug of reporter plasmid, 15 jg of effector plasmid, and 1 Rug of the f3-galactosidase expression plasmid pCH110 (15). CAT assays were performed as described by Gorman et al. (16). Gel Mobility Shift and Methylation Interference Assays. Electrophoretic mobility shift assays were performed as described by Singh et al. (17). For methylation interference assays, a 75-bp Hae III/BstNI fragment containing the NF-E1 binding site was subcloned into the HincIl site of pUC18. This plasmid was cut with BamHI, dephosphorylated with calf intestine phosphatase, and then labeled with [y32P]ATP by polynucleotide kinase. Partial methylation, binding reactions with Ag8 nuclear extract, and electrophoresis were performed as described (18). Screening of the cDNA Expression Library. A human B-cellderived Agtll cDNA library (human B-cell leukemia RPM
Immunoglobulin K gene expression is tissue specific and is developmentally regulated during B-cell development at the transcriptional level. Immunoglobulin K transcription is governed by two enhancers: one (EK) lies within the intron separating the joining and constant regions, and the other (KE3') lies downstream of the constant region (1-3). Both enhancers are tissue specific and both are developmentally controlled (3-6). That is, both enhancers are normally silent at the pre-B-cell stage but are active at the B-cell and plasma cell stages. The activity of the intron enhancer is controlled by the presence of the active form of NF-KB (7-10). The 3' enhancer, however, does not require NF-KB for its activity (6, 11). Therefore, the mechanism of its silence at the pre-B-cell stage is unclear. Silence of the KE3' enhancer at the pre-B-cell stage could be due to either the lack of positive-acting factors, the presence of negative-acting factors, or both. Previously, we functionally characterized the KE3' enhancer and identified a 132-base-pair (bp) core that retains 75% of the activity of the intact 1-kilobase (kb) enhancer in plasmacytoma cells (6). Sequences flanking this 132-bp core contribute very little to enhancer activity at this stage of B-cell development. It is unknown whether sequences within this core are capable of being active at the pre-B-cell stage of development or whether there are negative-acting elements in the KE3' enhancer. We report here that the KE3' enhancer 132-bp core is indeed active at the pre-B-cell stage. Downstream of the
Abbreviations: TK, thymidine kinase; CAT, chloramphenicol acetyltransferase. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M76541).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9804
Biochemistry: Park and Atchison
Proc. Natl. Acad. Sci. USA 88 (1991)
4265; Clontech) was screened with a labeled multimerized DNA binding site oligonucleotide according to the method of Vinson et al. (19). The oligonucleotide used for screening is GATCCTACCCCACCTCCATCTTGTTTGATA GATGGGGTGGAGGTAGAACAAACTATCTAG
DNA Sequence Analysis. DNA sequences were determined by the dideoxynucleotide chain-termination method on double-stranded DNA templates using the United States Biochemical sequencing kit. In Vitro Transcription and Translation. Clone 131 in pBluescript was linearized with Xba I and then transcribed with T7 polymerase using an mRNA capping kit according to the manufacturer's specifications (Stratagene). The capped mRNA was subjected to in vitro translation with a rabbit reticulocyte lysate (Promega).
RESULTS The E3' Core Is Active in pre-B Ceils and Is Flanked by Negative-Acting Elements. We set out to determine whether the 132-bp KE3' core is active at the pre-B-cell stage. The 132-bp core segment was linked to the herpesvirus TK promoter driving expression of the CAT gene. The activity of this construct was compared to that of the intact enhancer contained on a 728-bp DNA fragment (containing 311 bp 5' and 285 bp 3' of the core) after transfection into 1-8 pre-B cells. The 132-bp core segment was -10-fold more active than the 728-bp enhancer fragment in pre-B cells (Fig. 1A, compare constructs A and I). These results suggest the presence of negative regulatory elements within the 728-bp enhancer fragment that are deleted in the 132-bp fragment. To search for these negative elements, various deletion mutants were prepared (Fig. 1A). Deletion of sequences between positions 808 and 658 did not increase activity of the enhancer
A A
Retivo Activity
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FIG. 1. Identification of negative-acting segments in the KE3' enhancer. (A) DNA sequences present in each construct are shown on the left and are represented by open boxes. The relative activity of each construct in 1-8 pre-B cells is indicated on the right. Standard deviations were calculated from three to five transfection experiments. In construct J, x x indicates the position of a linker scan mutation in the NF-E1 binding site. (B) Position and orientation of sequences 523-808 inserted into construct H are indicated by arrows. CAT activities relative to construct H are shown.
9805
above that of the intact enhancer (Fig. 1A, compare constructs A, B, and C). However, progressive deletions to positions 615, 558, 536, and 523 (constructs D, F, G, and H, respectively) resulted in stepwise increases in CAT activity. Similar results were obtained with the pre-B-cell line 3-1 (data not shown). These results suggest the existence of multiple negative control elements that flank the 3' side of the 132-bp core of the KE3' enhancer. To ensure that the increased enhancer activity was not due to position effects caused by the various deletions and to determine whether the negative-acting segment was position or orientation independent, constructs K-N were prepared (Fig. 1B). Construct H was modified to contain sequences 523-808 on either the 3' side (K and L) or the 5' side (M and N) of the enhancer. All four constructs repressed enhancer activity, indicating that the negative-acting sequences are position and orientation independent (Fig. 1B). Repression ranged from 9- to 3.4-fold (11-29% of construct H). Identification of a Nuclear Factor That Binds to the NegativeActing Region of the ,cE3' Enhancer. To identify nuclear factors that bind to the negative-acting region of the KE3' enhancer DNA fragments were used as probes in electrophoretic mobility shift assays. A 75-bp Hae III/BstNI DNA fragment (residues 582-657) yielded a single bound complex with nuclear extracts prepared from cell lines representative of the pre-B-cell, B-cell, and plasma cell stages (data not shown). Unlabeled DNA fragments from the KE3' and the heavy-chain enhancers abolished protein-DNA interaction, but a DNA fragment from the K intron enhancer had no effect (data not shown). Oligonucleotides containing known immunoglobulin enhancer motifs were also used as competitors. An oligonucleotide containing the AE1 binding site abolished the DNA-nuclear factor interaction (Fig. 2 Upper), but not oligonucleotides containing the /ME2, IE3, ,uE4, puE5, NFKB, or octamer binding sites (data not shown). Thus, the nuclear factor that binds to the KE3' probe appears to be identical to the factor that binds to the immunoglobulin heavy-chain ME1 site. We therefore named this nuclear factor NF-E1. Dimethyl sulfate methylation interference assays indicated that methylation of G residues 623, 624, 626, 627, and 630 on the bottom DNA strand of the KE3' enhancer interfered with NF-E1-DNA interaction (Fig. 2 Left). These residues in the KE3' enhancer overlap with a region of 8 bp of identity with the heavy-chain ,uE1 site and closely correspond to sites identified by in vivo and in vitro ,E1 dimethyl sulfate footprinting (refs. 20 and 21; summarized in Fig. 2). Isolation and Characterization of cDNA Clones Encoding NF-El. A human B-cell cDNA library in Agtll was screened with an oligonucleotide containing the KE3' NF-E1 binding sequence by the method of Vinson et al. (19). Of 250,000 phage plaques screened, we obtained 3 positive clones designated 131, 324, and 1021. The largest cDNA clone (clone 324) was sequenced in its entirety and contains 2084 bp. Clone 131, although smaller than clone 324, extended the sequence an additional 92 bp in the 5' direction. The composite NF-E1 sequence spans 2176 bp and, beginning with the first ATG triplet at position 75, encodes an open reading frame of 414 amino acids (Fig. 3). The first methionine codon conforms well to the Kozak (22) consensus sequence. The termination codon is followed by 860 bp of 3' untranslated sequence. A polyadenylylation signal lies 4 bp upstream of the 3' end of the cDNA sequence. A second potential polyadenylylation signal lies 58 bp downstream of the translational termination codon. Analysis of the predicted amino acid sequence revealed a rather unusual structure. First, in the C-terminal portion are four zinc fingers of the Cis-Cis-His-His type. Second, the N-terminal portion of the protein is highly acidic with 21 of the first 53 (40%) amino acids being acidic. This region
9806
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Park and Atchison
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FIG. 2. NF-E1 also binds to the immunoglobulin heavy-chain
AE1 site. (Upper Right) The Hae III/BstNI DNA probe was assayed
by electrophoretic mobility shift assay with 3-1 pre-B-cell nuclear extract in the presence of 1 pmol of an unlabeled oligonucleotide containing the ,uE1 motif. Arrows B and F indicate the positions of the bound and free DNA probe, respectively. (Left) Dimethyl sulfate (DMS) methylation interference assay. Bound (B), free (F), and A+G sequencing reaction (A+G) lanes are shown. Protein contact sites are indicated by arrows. (Lower Right) Summary of the locations of these residues (open circles). DMS methylation interference pattern of the immunoglobulin heavy-chain 1LE1 site is included for comparison and is taken from Ephrussi et al. (20) and Weinberger et al. (21).
includes a portion with 11 consecutive acid residues between amino acids 43 and 53. The segment between residues 16 and 35 contains sequences consistent with the ability to form a negatively charged amphipathic a-helix. This highly acidic N terminus is reminiscent of transcriptional activation domains found in other transcriptional regulators (23, 24). Third, the segment between residues 70 and 80 is unusual in that it consists of 11 consecutive histidines. The transcriptional repressor Tst-1 (SCIP) also contains regions rich in histidine as well as a stretch of 6 consecutive histidine residues (25, 26). Fourth, the region between amino acids 154 and 198 is very rich in alanine and glycine residues (26 of 45; 58%). This region also includes a stretch of 4 consecutive serines between residues 164 and 167. The repressor Tst-1 (SCIP) also contains extensive regions rich in alanine and glycine (45%). In addition, the putative repressor domain in the transcniptional repressor Kruppel is rich in alanine residues (27). A search of the National Biomedical Research FoundationProtein Identification Resource (release 26) data base revealed homologies to numerous transcription factor IIIAtype finger proteins. Particularly striking was a 76% homology in the zinc finger region to the finger protein REX-i (28). No data base homologies to other segments of the NF-E1 sequence were observed.
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FIG. 3. DNA and predicted amino acid sequence of NF-E1. Amino acid and nucleotide coordinates are indicated on the right. Histidine residues are circled. Alanine and glycine residues are underlined. The two boxed regions denote the locations of the 11 consecutive acid residues near the N terminus and the zinc finger region at the C terminus. Asterisk shows the location of the termination codon. The 5' and 3' untranslated sequences are shown in italics. The two underlined sequences in the 3' untranslated region indicate potential polyadenylylation signals.
The sequences of clones 131 and 324 were compared over -800 bp. Both sequences were identical except at two locations. At nucleotide position 268, a G to A transition results in an arginine to histidine change in clone 324. In addition, a CCA codon is deleted in clone 324 between nucleotide positions 271 and 273, resulting in the loss of a single histidine residue. These differences presumably reflect polymorphisms of the same gene. DNA Binding Characterization of the Cloned and Endogenous NF-El Proteins. Clone 131 was transcribed and translated in vitro and the synthesized protein was assayed for binding to the KE3' NF-E1 DNA probe. The in vitro synthesized protein bound to the KE3' NF-E1 probe and migrated with a mobility identical to the endogenous protein (Fig. 4A, lanes 2 and 3). The protein-DNA complex was abolished by competition with unlabeled oligonucleotides containing the KE3' NF-E1 or the immunoglobulin heavy-chain ,E1 binding sites, but not with one containing a mutant NF-E1 sequence (lanes 4-6). The zinc chelator 1,10-phenanthroline (29) abolished DNA binding by the in vitro synthesized and the endogenous proteins (Fig. 4A, lane 7; Fig. 4B, lanes 1 and 2).
Biochemistry: Park and Atchison
Proc. Natl. Acad. Sci. USA 88 (1991)
in the opposite orientation (GAL4-rEl) or the GAL4 1-147 sequences alone (GAL4 1-147) had no effect on expression (lanes 2 and 4). The above repression could be due to NF-E1 directly repressing promoter activity. Alternatively, NF-E1 could be titrating factors necessary for promoter activity (squelching). To distinguish between these possibilities, we cotransfected the various effector constructs with a reporter plasmid that lacked the GAL4 DNA binding site (TKCAT). TKCAT expression was unaffected by cotransfection with the GAL4-El construct, indicating that DNA binding is necessary for the repressive activity of NF-E1 (Fig. 5, lanes 5-8). To ensure that the repression observed above was not due to an unusual feature of the GAL4-El chimera, we expressed the native NF-E1 cDNA without GAL4 sequences. The NF-E1 expression plasmid (SV-E1) repressed activity of a TKCAT reporter containing the NF-E1 binding site, (E1)4TKCAT, while an expression plasmid with the NF-E1 sequence in the opposite orientation (SV-rE1) had no effect (lanes 9-11). To assess the functional role of the NF-El binding site in the context of the intact KE3' enhancer, we prepared a linker scan mutation of the NF-El binding site. This construct gave a small, but reproducible, 1.3-fold activation of the intact KE3' enhancer in pre-B cells (Fig. 1A, construct J). This low level of activation was not unexpected given our evidence that the KE3' enhancer contains multiple negative elements (Fig. 1A). Our results are consistent with the NF-E1 site playing a negative role in KE3' enhancer activity rather than a positive role.
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