A Short Conserved Motif Is Required for Repressor Domain Function in the Myeloid-specific Transcription Factor CCAAT/Enhancer-binding Protein epsilon *

Nicholas D. Angerer, Yang Du, Demet Nalbant, and Simon C. WilliamsDagger §

From the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center and Dagger  Southwest Cancer Center at University Medical Center, Lubbock, Texas 79430

    ABSTRACT
Top
Abstract
Introduction
References

CCAAT/enhancer-binding protein epsilon  (C/EBPepsilon ) is expressed almost exclusively in the myeloid lineage of the hematopoietic system and functions during terminal differentiation of neutrophils and macrophages, and in the regulation of cytokine gene expression in macrophages and T cells. We have undertaken a series of structure/function studies on the murine C/EBPepsilon polypeptide to investigate the mechanism by which C/EBPepsilon activates transcription. Studies with deletion mutants and fusion proteins consisting of C/EBPepsilon sequences joined to the Gal4 DNA-binding protein identified two transcriptional activation domains in C/EBPepsilon . Removal of sequences between the two activation domains or sequences between the second activation domain and the C-terminal DNA binding domain significantly increased the activity of C/EBPepsilon , suggesting the presence of two separate regulatory domains (designated RD-1epsilon and RD-2epsilon ). RD-1epsilon behaved as a classic active repressor domain being capable of inhibiting adjacent activation domains irrespective of their origin and when linked to a heterologous DNA binding domain. Mutagenesis studies revealed a short motif in RD-1epsilon that appears to be a target site for protein-protein interactions and is conserved in repressor domains from C/EBPbeta , Sp3, c-Fos, and FosB. The juxtaposition of activation and repressor domains may permit C/EBPepsilon to function as a transcriptional activator or repressor at different stages of myeloid differentiation or as an inducible transcriptional activator of cytokine genes.

    INTRODUCTION
Top
Abstract
Introduction
References

Transcription factors play important roles in the commitment of precursor cells to particular hematopoietic lineages and the terminal differentiation of specific cell types (1). The CCAAT/enhancer-binding protein (C/EBP)1 family of basic region/leucine zipper transcription factors consists of six members that display similar DNA binding specificities and are capable of homo- and heterodimerization in all combinations (reviewed in Ref. 2). Four family members, C/EBPalpha , C/EBPbeta , C/EBPdelta , and C/EBPepsilon , are expressed in myeloid cells and appear to play different roles in differentiating and mature cells (3, 4). C/EBPalpha appears to be critical for granulocytic differentiation (5), whereas C/EBPbeta and C/EBPdelta appear to function primarily as regulators of cytokine gene expression during inflammatory responses in macrophages (6-8). C/EBPepsilon was first identified based on its relatedness to C/EBPalpha (9) and is almost exclusively expressed in myeloid cells, with additional sites of expression in lymphoid cells and ovary (10-12). C/EBPepsilon expression is essentially undetectable in uncommitted hematopoietic progenitors but increases during myeloid differentiation resulting in high level expression in mature neutrophils and macrophages (12-14). Mice lacking C/EBPepsilon display defects in granulocytic development (15), a phenotype that is also seen in C/EBPalpha -deficient mice (5). C/EBPepsilon appears to act later in granulopoiesis than C/EBPalpha , and its expression may be activated by C/EBPalpha in this lineage (16, 17).

Although the primary defects observed in C/EBPepsilon -deficient mice were in the development of the granulocyte lineage, there is evidence that C/EBPepsilon also functions in other hematopoietic cell types. C/EBPepsilon mRNA is present in primary murine macrophages and in multiple immortalized monocytic and macrophage cell lines, and a 34,000 molecular weight C/EBPepsilon polypeptide has been detected in the IC-21 P388D1(IL-1) cell line (12). Ectopic expression of C/EBPepsilon in a lymphoid cell line activated the expression of a number of genes normally expressed in macrophages, including lipopolysaccharide-regulated cytokine genes and the macrophage-CSFR (M-CSFR) gene (12). The lack of defects in monocytic development and function in C/EBPepsilon -deficient mice may be due to functional redundancy among C/EBP family members expressed in this cell lineage (7). C/EBPepsilon mRNA was also detected in the human Jurkat T cell line (10), and the expression of some genes normally expressed in T cells, including interleukin-2 and -4, was diminished or abolished in C/EBPepsilon -deficient mice (15).

The C/EBPepsilon gene contains at least one intron and alternative splice sites and promoters permit the production of multiple polypeptides in humans (14). We have detected a single C/EBPepsilon mRNA in rodent cells that may direct the synthesis of two forms of C/EBPepsilon due to the presence of at least two in frame translation initiation codons (12). We have carried out a structure/function analysis of the murine C/EBPepsilon polypeptide to identify domains responsible for the transcriptional activity of C/EBPepsilon . We have identified two activation domains in C/EBPepsilon , one at the N terminus and a second close to the center of the protein. In addition, two regions within the C/EBPepsilon polypeptide act as repressor domains and are likely to be sites of protein-protein interactions that modulate the activity of C/EBPepsilon .

    EXPERIMENTAL PROCEDURES

Plasmid Construction-- The murine C/EBPepsilon mRNA contains two in frame initiation codons separated by 96 base pairs (12). PCR was used to introduce NcoI sites at the first of these two sites and a HindIII site following the termination codon, and the resultant cDNA was inserted into the pMEX eukaryotic expression vector (9). N-terminal and internal deletion constructs were prepared by PCR in a manner similar to that previously described for C/EBPbeta (18). Briefly, PCR was used to introduce either BglII or BamHI restriction sites at specific positions within the C/EBPepsilon coding sequence, and the resultant fragments were combined to generate the required recombinant. The sequences of the oligonucleotides used in this study are shown in Table I. Each was designed to insert restriction sites at or just following a proline residue and each carried a G-rich sequence at the 5' end to enhance enzyme digestion.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Oligonucleotides used in the synthesis of C/EBPvarepsilon segments
Oligonucleotide primers were synthesized with a G/C-rich clamp sequence preceding the restriction site to be inserted. Each 5'-oligonucleotide contained an internal BglII site. Each 3'-oligonucleotide contains an internal BamHI site, and the amino acid specified at the beginning of each 5'-oligonucleotide or end of each 3'-oligonucleotide is indicated.

Gal4 fusion genes were constructed in the Gal0 plasmid, which is similar to pSG424 (19). This plasmid encodes amino acids 1-147 of the yeast Gal4 transcriptional activator located downstream of the SV40 early promoter. All C/EBPepsilon - and VP16-derived segments were inserted downstream of the Gal4 sequences as NcoI/ClaI fragments as described previously (18). The VP16 activation domain fragment used here encodes amino acids 429-456 (20). Gal4 C/EBPalpha -(1-108), Gal4 C/EBPbeta -(1-83), and Gal4 C/EBPepsilon -(33-64) (previously named Gal4 CRP1-1-32)) encode the indicated amino acids from each C/EBP protein and have been described previously (18). The C/EBP-dependent (DEI)4-35AlbLUC (which consists of four copies of the DEI element from the serum albumin gene linked to the minimal promoter region of the same gene) and Gal4-dependent G5E1bLUC (which consists of five copies of a Gal4-binding site upstream of the E1b minimal promoter) luciferase reporter plasmids were described previously (18).

Cell Culture and Transfections-- The human HepG2 hepatocarcinoma and monkey COS-1 kidney cell lines were cultured in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VI) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). HepG2 cells were transfected at approximately 30-40% confluency in 3.5-cm dishes using a standard calcium phosphate co-precipitation method (18). In a typical experiment 2 µg of reporter and 2.5-5 µg of effector plasmid of pMEX-based constructs or 2 µg of reporter and 0.6 µg of effector plasmid for Gal4-based constructs were used. DNA concentrations were equalized using pBluescript (Stratagene). Although HepG2 cells do not express their endogenous C/EBPepsilon gene, we have obtained similar transactivation results in multiple cell lines, including some that do express C/EBPepsilon .2 Consequently, HepG2 cells were used in these studies as they are relatively easy to transfect and yield consistent results. However, we were unable to detect Gal4 proteins in transfected HepG2 cells and used COS-1 cells instead for this purpose. COS-1 cells were transfected by electroporation as described.3 Statistical analyses were carried out using a two-tailed Student's t test, and results that were statistically significant at 95% or 99% confidence levels are indicated in the figures. Luciferase assays were carried out as described (12).

Site-directed Mutagenesis-- Point mutations were introduced into the C/EBPepsilon coding sequence using the Gene Editor kit (Promega) according to the manufacturer's instructions. Mutagenic oligonucleotides were synthesized carrying 15 base pairs on either side of the mutation site, and a restriction site was generally included to aid in analysis. In all mutagenic reactions the target residue was changed to an alanine, and introduction of mutations was verified by sequencing. The names and sequence of oligonucleotides utilized were as follows (base changes are indicated in lowercase): A82/4 changes Pro82, Ala83, and Asp84 to (Ala)3, 5'-AAATGGCCGAGGcgCtGCaGcCAGGTAGTGAGG-3'; A121/3 changes Lys121, Glu122, and Glu123 to (Ala)3, 5'-GCTGTGGCGGTGgctGcaGcGCCTCGAGGGCCA-3'; A121/2 changes Glu122 and Glu123 to (Ala)2, 5'-GTGGCGGTGAAGGccGcGCCTCGAGGGCCA-3'; and Ala123 changes Glu123 to Ala, 5'-GCGGTGAAGGAGGcGCCTCGAGGGCCA-3'. The A121/2 mutation which changes Lys121 and Glu122 to (Ala)2 was generated by repairing the third mutated position in A121/3 by PCR using the Glu128 oligonucleotide shown in Table I.

Nuclear Extract Preparation and Western Blotting-- For transfected HepG2 cells we found that most nuclear proteins were present in the pellet after centrifugation of the extracts prepared for luciferase reactions. These were dissolved in Laemmli sample buffer, and equivalent aliquots of each reaction were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Micron Separations, Westborough, MA). Nuclear extracts were prepared from COS-1 cells as described previously (8). Immune detection was carried out as described previously (22) using either a C/EBPepsilon -specific rabbit polyclonal antiserum (C-22, Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal antiserum directed against the DNA binding domain of Gal4 (RK5C1, Santa Cruz Biotechnology). Immune complexes were detected using the Supersignal chemiluminescence detection kit (Pierce).

    RESULTS

The C/EBPepsilon Protein Contains Two Regions That Function as Transcriptional Activation Domains-- To identify functional domains that mediate transcriptional activation by C/EBPepsilon , we compared the ability of wild type C/EBPepsilon and two proteins lacking N-terminal segments to activate the (DEI)4-35AlbLUC reporter construct in HepG2 human hepatocarcinoma cells (Fig. 1A). This construct was activated 45-fold by full-length C/EBPepsilon ; however, deletion of either 96 or 139 N-terminal amino acids decreased transcriptional activity to background levels. Thus, C/EBPepsilon is capable of functioning as a potent transcriptional activator, and the N-terminal portion of C/EBPepsilon contains most or all of the activation sequences. The N-terminal sequences of C/EBPepsilon , C/EBPalpha , C/EBPbeta , and C/EBPdelta possess three segments of significant sequence similarity that correspond to three activation domain modules (ADM) first identified in C/EBPbeta (18) (Fig. 1B). We previously demonstrated that amino acids 33-64 of C/EBPepsilon (which contain ADM2 and -3) functions as a relatively weak activation domain when fused to the DNA binding domain of the yeast transcriptional activator protein Gal4 (18). Therefore, we tested whether inclusion of the first 32 amino acids of C/EBPepsilon , which contain sequences similar to the ADM1 domain of C/EBPbeta , would increase the potency of the N-terminal C/EBPepsilon activation domain (ADI). The addition of amino acids 1-32 resulted in a slight but significant (p < 0.01) increase in C/EBPepsilon AD activity (compare Gal4 epsilon -(33-64) to Gal4 epsilon -(1-64)), indicating that the ADM1 region is likely to be a functional component of ADI of C/EBPepsilon (Fig. 1C). In comparison, the equivalent regions of the C/EBPalpha (amino acids 1-108) and C/EBPbeta -(1-83) proteins were 4.5- and 7.5-fold, respectively, more powerful in this assay. The C/EBPepsilon polypeptide also shares a second region of homology (amino acids 140-162) with C/EBPalpha and C/EBPdelta which includes sequences identified as a second activation domain in C/EBPalpha (23-25). This region of C/EBPepsilon functioned as a weak activation domain (referred to as ADII) when fused to the Gal4 DBD (Fig. 1C).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   C/EBPepsilon contains two transcriptional activation domains. A, full-length C/EBPepsilon -(1-281) and two N-terminal deletion mutants initiating at amino acids 97 and 141 were co-transfected into HepG2 cells with the (DEI)4-35AlbLUC reporter plasmids. Luciferase activities were measured and are represented graphically as fold activation (±S.E.) compared with the reporter plasmid alone. The structure of the C/EBPepsilon proteins is diagrammed, and shaded regions represent sequence similarity to two activation domains (ADI and ADII) previously identified in C/EBPalpha and C/EBPbeta . B, the N-terminal sequences of C/EBPepsilon , C/EBPalpha , C/EBPbeta , and C/EBPdelta were aligned to identify regions of similarity. Amino acid coordinates are shown at the ends of each sequence, and identical amino acids are represented with vertical bars and conservative substitutions with colons. Three distinct regions of similarity first identified in C/EBPbeta (named activation domain modules (ADM) 1, 2, and 3) are indicated. C, Gal4 fusion proteins containing the indicated C/EBPepsilon coding sequences were tested for their ability to activate a Gal4-dependent reporter construct in HepG2 cells. Gal4 proteins containing ADI regions from C/EBPalpha and C/EBPbeta were included for comparison and ADM1, -2, and -3 regions are shaded in each. The activity of each protein is shown as fold activation compared with the reporter plasmid alone.

C/EBPepsilon Contains Two Internal Regulatory Domains-- We next tested whether the C/EBPepsilon polypeptide contains additional sequences outside the activation domains that modulate its transcriptional activity. C/EBPepsilon proteins lacking internal sequences were tested for their ability to activate (DEI)4-35AlbLUC in HepG2 cells (Fig. 2A). The removal of amino acids 65-194 (i.e. all sequences between ADI and the DBD) resulted in a highly significant (p < 0.01) 6-fold stimulation of transcriptional activity (compare 1-281 and 1-281 (Delta 65-194)). Proteins lacking sequences between ADI and ADII (1-281(Delta 65-96) and 1-281(Delta 65-139), Fig. 2A) were also more active than the wild type C/EBPepsilon protein in this assay, approximately 4- and 7-fold, respectively, identifying a region between amino acids 65 and 140 (termed regulatory domain-1epsilon or RD-1epsilon ) that negatively regulates C/EBPepsilon activity. Removal of amino acids between ADII and the DBD did not significantly affect C/EBPepsilon activity (1-281(Delta 163-194) Fig. 2A), but combining the deletion of amino acids 163-194 with the deletion of the RD-1epsilon region resulted in further stimulation of C/EBPepsilon activity. For example, a protein lacking amino acids 65-139 and 163-194 displayed approximately 1.5-fold greater activity than a protein lacking amino acids 65-139 alone (Fig. 2A), and similar results were observed with other constructs lacking sequences in both regions (data not shown). We conclude that a second negatively acting domain, named RD-2epsilon , is located between amino acids 163 and 194 which in this context appears to function in combination with RD-1epsilon . Similar steady-state levels of each recombinant protein were detected by Western analysis (Fig. 2B) indicating that the observed effects were not simply due to differences in protein expression levels or stability.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Identification of two regulatory domains in C/EBPepsilon . A, a series of C/EBPepsilon expression vectors lacking internal sequences were constructed in pMEX and were tested for their ability to activate (DEI)4-35AlbLUC in HepG2 cells. The constructs are named according to the amino acids removed in each case, and their activities are represented graphically as fold activation compared with the reporter plasmid alone. Activation and DNA binding domains are labeled on top, and the approximate location of two regulatory domains identified by these studies (RD-1epsilon and RD-2epsilon ) are indicated below the figure. The actual activity value for each construct shown at the end of each bar represents the average of at least three independent transfections. Activities that were significantly different from the wild type protein are indicated at 95 (*) and 99% (**) confidence levels. B, representative Western blot analysis showing comparable protein levels expressed from each construct in nuclear extracts of transfected HepG2 cells.

Fine Mapping of Regulatory Domains Using Gal4 Fusion Proteins-- To test whether C/EBPepsilon regulatory domains could function when attached to a heterologous DNA binding domain, we constructed a series of Gal4-C/EBPepsilon chimeric genes and tested their ability to activate expression from the Gal4-dependent G5E1bLUC reporter plasmid in HepG2 cells. Fusion proteins bearing the first 64, 98, or 116 amino acids of C/EBPepsilon all functioned as strong transcriptional activators (Fig. 3A). However, a significant 34-fold decrease (compared with Gal4 epsilon -(1-64)) in activity was observed when C/EBPepsilon sequences were extended to amino acid 128, indicating that RD-1epsilon is located N-terminal to this amino acid. A 5-fold increase in activity was observed upon inclusion of sequences up to amino acid 162, presumably due to the inclusion of ADII, and a further decrease (3.3-fold) occurred when the C/EBPepsilon sequences were extended to amino acid 193 to include the RD-2epsilon -containing region. Finally, we tested whether RD-2epsilon is capable of functioning in the absence of RD-1epsilon by fusing amino acids 162-193 directly to ADI. The addition of this region resulted in a 4-fold decrease in activity (compare Gal4 epsilon -(1-64) to Gal4 epsilon -(1-64)(162-193) indicating that RD-2epsilon is capable of repressing ADI function in a position- and RD-1epsilon -independent fashion in Gal4 fusions. Unfortunately, we were unable to detect expression of Gal4 fusion proteins in nuclear extracts of HepG2 cells; however, they were detectable after transient transfection of COS-1 cells. All Gal4 fusions behaved similarly in transactivation assays in COS-1 cells2 and were expressed at similar levels (Fig. 3B) again indicating that the observed functional differences are not due to variations in protein levels.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   RD-1epsilon and RD-2epsilon repress AD function independent of DNA binding. A, various segments of the C/EBPepsilon coding sequence were attached to the DNA binding domain from the yeast transcriptional activator protein Gal4, and the resultant chimeric proteins were tested for their ability to activate transcription from the G5E1bLUC reporter plasmid. The name of each construct refers to the coordinates of the C/EBPepsilon sequences included in each protein, and the activity of each protein is listed numerically as fold activation compared with the reporter plasmid alone. A schematic representation of C/EBPepsilon is shown with functional domains labeled. The repression index was calculated by dividing the activities of proteins that differ by the presence of RD-1epsilon and/or RD-2epsilon . Transfections were repeated at least four times for each construct, and values that were significantly different at the 99% confidence level are indicated (**). B, Western blot analysis of a subset of Gal4-C/EBPepsilon fusion proteins expressed in COS-1 cells.

RD-1epsilon and RD-2epsilon Repress a Heterologous Activation Domain-- To study the activation domain specificity of the two regulatory domains in C/EBPepsilon , we tested their ability to repress the activity of Gal4 fusion proteins containing the potent activation domain from the herpes simplex virus VP16 protein (Fig. 4A). Initially we tested whether C/EBPepsilon sequences could repress transactivation when placed downstream of the VP16AD, i.e. in a similar position relative to the activation domain as in C/EBPepsilon . The activity of the Gal4VP16 protein on the G5E1bLUC reporter plasmid in HepG2 cells was set at 100%, and the activity of each fusion protein was calculated relative to this value. The inclusion of amino acids 64-98 or 64-116 did not diminish the activity of the VP16AD. However, a protein containing amino acids 64-128 displayed only 16% of the activity of the control, indicating that RD-1epsilon is capable of repressing the activity of the VP16AD. To map the N-terminal boundary of RD-1epsilon , three additional constructs containing C/EBPepsilon sequences initiating at amino acid 97 were tested. Fusion proteins carrying amino acids 97-162 or 97-193 were 55 and 66% as active as Gal4VP16AD; however, the attachment of amino acids 97-128 repressed VP16AD activity to 4% of control levels. These data further refine the position of RD-1epsilon between amino acids 97 and 128, and the higher activity of constructs containing amino acids 97-162 and 97-193 is likely due to the presence of ADII. In the final construct in this series, RD-2epsilon (amino acids 162-193) was attached to the C terminus of the VP16AD. This protein displayed only 27% (p < 0.01) of the activity of the control protein, indicating that RD-2epsilon was also capable of inhibiting a heterologous AD. Western blotting confirmed that all proteins were expressed at comparable levels (Fig. 4B, for example).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4.   RD-1epsilon and RD-2epsilon are capable of repressing activity of a heterologous activation domain. A, a series of Gal4 fusion proteins were constructed to test whether the regulatory domains from C/EBPepsilon could inhibit the activity of the heterologous activation domain from the viral transactivator protein VP16, which is represented by the hatched box. Each fusion was generated in two forms that differed in the relative orientation of the VP16 and C/EBPepsilon sequences, and the C/EBPepsilon amino acids are indicated in parentheses in the construct names. Each protein was tested for its ability to activate G5E1bLUC in HepG2 cells, and activities are listed relative to the activity of Gal4-VP16, which was set at 100%. The activities represent the average results of at least three independent transfections. Protein activities that are significantly lower than that of Gal4VP16AD at the 95 (*) or 99% (**) confidence levels are indicated. These data demonstrate that the minimal RD-1epsilon sequence is located between amino acids 97 and 128 (indicated with a hatched box), and RD-2epsilon is located between amino acids 162 and 193 (indicated with a stippled box). B, representative Western blot showing comparable accumulated levels of proteins with significantly different activities.

A second series of chimeric genes was constructed in which the order of the C/EBPepsilon and VP16 sequences was reversed to test for position-dependent effects. Although some differences in the repressive activity of the C/EBPepsilon regulatory domains were observed in the two sets of proteins, the overall pattern of activity was similar (Fig. 4A). The minimal RD-1epsilon and RD-2epsilon segments (amino acids 97-128 and 162-193, respectively) both repressed VP-16AD function, albeit to a slightly lesser but statistically significant (p < 0.01) extent than in the previous set of chimeras. In this second arrangement, RD-2epsilon appeared to be an efficient repressor module even in the presence of ADII of C/EBPepsilon (compare Gal4 epsilon -(97-162)-V to Gal4 epsilon -(97-193)-V)), which could potentially be explained by its close proximity to the VP16AD in these proteins.

RD-1epsilon Contains Sequence Motifs That Are Conserved in Other Repressor Domains-- We next compared the sequence of amino acids 64-128 of C/EBPepsilon to the sequence of repressor domains identified in other transcription factors. We identified two small conserved motifs (termed conserved motif (CM) 1 and 2 in Fig. 5) in a subset of factors examined. CM1 is located between amino acids 82 and 86 of C/EBPepsilon and is conserved in C/EBPepsilon , rat and human C/EBPbeta , and Sp3 but is absent in chicken C/EBPbeta , c-Fos, and FosB. A consensus sequence was derived for CM1 as Pro-Ala-Asp-X-B, where X is any amino acid and B is a basic amino acid. CM1 lies outside the region defined as RD-1epsilon and would not be predicted to be critical for RD-1epsilon function. CM2 is located between amino acids 121 and 128 of C/EBPepsilon (i.e. within RD-1epsilon ), and related sequences were present in all proteins listed. CM2 has the consensus sequence B-Glu-Glu-X-X-X-Pro-Glu and is most highly conserved in C/EBPepsilon , C/EBPbeta (human and chicken), Sp3, and FosB.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   C/EBPepsilon shares two conserved motifs with regulatory domains from other transcription factors. The sequence of regulatory domains from rat C/EBPepsilon (this study), rat C/EBPbeta (RD1 (18)), human C/EBPbeta (46), chicken C/EBPbeta (21), Sp3 (31), Fos and FosB (ID1 (32)) were aligned to identify conserved amino acids. Conserved residues in two motifs, named CM1 and CM2, are shown in reverse print. The amino acid coordinates are shown for each sequence, and the consensus sequence for each motif is shown below the alignment. B, basic amino acid (Lys or Arg); X, any amino acid.

We next tested whether mutating residues in CM1 and/or CM2 would diminish RD-1epsilon function. The mutations focused on the conserved PAD and KEE motifs in CM1 and CM2, respectively (Fig. 5). Both motifs were replaced with alanines in one or both of the Gal4 epsilon -(1-128) and Gal4 epsilon -(64-128)-V fusion proteins (see Figs. 3 and 4). Mutation of the CM1 sequence had no effect on the activity of Gal4 epsilon -(1-128) as proteins thus configured displayed repressed activities similar to the wild type protein (A82/4 mutant in Fig. 6A). However, mutating CM2 to three alanine residues (A121/3 mutants) resulted in significant increases in the activity of both proteins, 12-fold in Gal4 epsilon -(1-128) and 29-fold in Gal4 epsilon -(64-128)-V (Fig. 6, A and B). Mutating pairs of amino acids (A121/2 and A122/3) or the terminal glutamic acid residue alone (A123) all significantly reduced the activity of the regulatory domain in Gal4 epsilon -(1-128) and, where tested, Gal4 epsilon -(64-128)-V indicating that residues 121 and 123 within CM2 are critical for RD-1epsilon function. The contribution of the glutamic acid at position 122 remains to be verified. Equivalent levels of each protein were detected by Western blotting (Fig. 6, C and D).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6.   CM2 is required for RD-1epsilon function. Conserved residues in CM1 and CM2 were changed to alanine in the Gal4 epsilon -(1-128) (A) and Gal4 epsilon -(64-128)-V (B) constructs. Each protein was tested for its ability to activate the G5E1bLUC reporter plasmid in HepG2 cells. The activities of proteins carrying the indicated mutations are shown relative to each wild type construct and represent the average of three independent transfections. Statistical analysis is represented as in Fig. 2. NT, not tested. C and D, representative Western blot of recombinant proteins.

RD-1epsilon Appears to be a Site for Protein-Protein Interactions-- Finally, we tested whether co-expression of RD-1epsilon might result in derepression of a Gal4-C/EBPepsilon fusion protein by competing for binding of a nuclear protein. Increasing amounts of the C/EBPepsilon -(97-281) expression vector were co-transfected with Gal4 epsilon -(1-128) into HepG2 cells, and luciferase activities were measured (Fig. 7). At the highest level of C/EBPepsilon -(97-281) expression vector the activity of Gal4 epsilon -(1-128) was 3-fold higher than in the absence of any competitor molecule. To test whether this effect was attributable to the co-expression of a molecule containing a functional RD-1epsilon element, a second series of transfections were carried out using a competitor molecule carrying the A121/3 mutation, which changes the KEE motif to three alanine residues and abolishes RD-1epsilon activity (see Fig. 6, A and B). In this case relief of inhibition was not observed, in fact luciferase activities decreased as the amount of competitor was increased which we interpret to be the result of competition between the promoters in the two expression vectors. Both proteins were efficiently synthesized as measured by Western analysis (Fig. 7B). Taken together, the results of these assays indicate that a co-expressed RD-1epsilon -containing protein is able to relieve inhibition in Gal4 epsilon -(1-128) presumably by competing for a cellular protein that normally binds to RD-1epsilon and mediates the repressive effect of this domain.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7.   Co-expression of RD-1epsilon -containing C/EBPepsilon deletion mutant relieves RD-1epsilon -mediated repression of a Gal4 fusion protein. A, HepG2 cells were transfected with G5E1bLUC and an expression vector encoding Gal4 epsilon -(1-128) in the absence or presence of increasing amounts of N-terminal C/EBPepsilon deletion mutants encoding amino acids 97-281. Two competitors were used, a wild type protein carrying the KEE motif at amino acids 121-123 (black bars) and a mutant (A121/3) bearing three alanines in place of this motif (white bar). Luciferase activities were measured and are expressed relative to the activity of Gal4 epsilon -(1-128) alone which was set at 1.0. The results represent the averaged values (± S.E.) of a representative experiment performed in triplicate. B, Western blot analysis of nuclear proteins from transfected cells, probed with an anti-C/EBPepsilon antiserum. The position of the two competitor proteins is indicated.


    DISCUSSION

The individual members of the C/EBP family possess a highly conserved bZIP DNA binding domain and are consequently capable of binding to identical recognition sequences in the control regions of target genes (9, 26). Therefore, the unique functions of each protein are likely to be controlled by domains outside the DNA contacting basic region, and numerous structure/function studies have been carried out to identify these domains. In the case of C/EBPepsilon , we have previously identified domains that control DNA binding, dimerization (9), nuclear localization (27), and transcriptional activation (12). To understand more clearly the activity of C/EBPepsilon is regulated we have now identified additional domains that control the activity of C/EBPepsilon . In addition to defining sequences that function as transcriptional activation domains, we also identified two separate regulatory domains, RD-1epsilon and RD-2epsilon , that repress C/EBPepsilon activity. RD-1epsilon is located between the two transcriptional activation domains and is wholly contained in the region spanning amino acids 97-128. RD-2epsilon is located N-terminal to the DNA binding domain, and its activity in the context of C/EBPepsilon was only evident when RD-1epsilon was also absent. However, RD-2epsilon was capable of independently inhibiting a linked activation domain in Gal4 fusion proteins. Over the past several years, numerous nuclear proteins have been identified that contain domains that negatively regulate transcription (reviewed in Ref. 28). Two criteria have been used to define these domains as active repressor domains as follows: the ability to function in a modular fashion when attached to a heterologous DNA binding domain, and second to act as the site for interactions with corepressor proteins (29). By these criteria, RD-1epsilon behaves as an active repression domain as it inhibited the activity of C/EBPepsilon and VP16 activation domains when attached to the Gal4 DBD. In addition, our competition data indicate that RD-1epsilon is a site for protein-protein interactions. RD-2epsilon was also capable of inhibiting a linked activation domain when attached to a heterologous DNA binding domain thereby satisfying one criterion for characterization as an active repression domain.

Specific motifs within repressor domains, often comprised of just a few amino acids, have been identified that are critical for repressor domain function. For example, the repressor domains of the Drosophila proteins Hairy and Enhancer of Split contain the four amino acid motif, Trp-Arg-Pro-Trp (WRPW), which mediates interactions with members of the Groucho family of corepressors (30). Our mutation studies identified the sequence Lys-Glu-Glu (KEE) in RD-1epsilon as being required for repressor domain function and for protein-protein interactions. This same motif is required for activity of the repressor domain of Sp3 (31), and similar sequences are present in RD-1 of C/EBPbeta (18) and the inhibitory domain (ID-1) of c-Fos and FosB (32). The corresponding amino acids have not been mutated in C/EBPbeta , c-Fos, or FosB; however, the repressor domain of c-Fos has been shown to be a site for protein-protein interactions (32). Therefore, KEE or a similar motif may be a signature for repressor domains from proteins derived from diverse transcription factor families. The KEE motif is not present in RD-2epsilon ; however, as shown in Fig. 8, RD-2epsilon contains multiple copies of the consensus minimal recognition sequence for members of the MAP kinase family, the dipeptide (Ser/Thr)-Pro (33). Therefore, it is conceivable that phosphorylation of the RD-2epsilon element by a member of the MAP kinase family might regulate the activity of C/EBPepsilon . Little is currently known concerning the phosphorylation of C/EBPepsilon ; however, support for this hypothesis comes from studies of other C/EBP family members, particularly C/EBPbeta . Comparison of this region of RD-2epsilon to the corresponding regions of C/EBPalpha , C/EBPbeta , and C/EBPdelta (i.e. the region immediately N-terminal to the basic region) reveals multiple putative MAP kinase sites in each protein (Fig. 8). Phosphorylation of the threonine residue at position 238 of human C/EBPbeta (amino acid 168 of rat C/EBPbeta ) via a Ras-dependent MAP kinase pathway in NIH3T3 and P19 cells has been shown to stimulate C/EBPbeta activity (34). In addition, mutation of this residue to a non-phosphorylatable residue decreased C/EBPbeta activity in P19 and neuronal cell lines (34, 35). Mutagenesis studies should begin to define the importance of this region for RD-2epsilon and C/EBPepsilon activity.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   Putative MAP kinase sites in RD-2epsilon are conserved in C/EBPalpha , C/EBPbeta , and C/EBPdelta . Amino acids 174-194 from RD-2epsilon were aligned with the corresponding region of three other C/EBPs in order to demonstrate the presence of multiple copies of the dipeptide (Ser/Thr)-Pro (underlined) in each protein. The threonine residue at position 168 of C/EBPbeta can be phosphorylated by MAP kinase (34), and it is possible that each C/EBP protein could be targeted by members of this kinase superfamily.

Two previous studies proposed that the activity of C/EBPbeta was inhibited by intermolecular interactions between the repressor and activation domains, which served to block access of the activation domain to its target proteins (18, 36). However, the data presented here suggest that the RD-1epsilon , and probably the RD-2epsilon , repressor domains of C/EBPepsilon act as sites for interactions with accessory proteins that act as corepressors. Multiple proteins have been identified that physically interact with members of the C/EBP family, although most of these proteins interact via the bZIP domain. These proteins are generally transcriptional activators that bind to adjacent sites on promoters and synergize with C/EBP proteins in activating transcription of the target gene and include NFkappa B (37, 38), AML1, and PU.1 (39), glucocorticoid receptor (40), Fos and Jun (41), Nopp140 (42), and Sp1 (43). In addition, a limited number of proteins have been identified that interact with regions outside the DNA binding domain of C/EBP proteins, including Sp1 (43) and the retinoblastoma protein (44, 45); however, it is unlikely that any of these proteins interact with RD-1epsilon . The identification of the KEE motif as a critical component of RD-1epsilon should facilitate the identification of its cognate binding proteins and provide clues as to its function in C/EBPepsilon .

Two alternative scenarios might explain the role of these repressor domains in determining the biological function of C/EBPepsilon . First, interaction of corepressors with the repressor domains may serve to maintain C/EBPepsilon in an inactive state until an activating signal is received. This model would explain the ability of C/EBPepsilon to participate in the inducible activation of cytokine genes upon lipopolysaccharide stimulation of macrophages and other cell types (12). Further studies will determine whether phosphorylation of potential MAP kinase sites in RD-2epsilon are involved in regulating C/EBPepsilon activity. Alternatively, it is becoming apparent that transcriptional repression may be just as important as activation in regulating cell-specific gene expression during development. Therefore, C/EBPepsilon could play dual regulatory roles as a transcriptional activator and repressor, depending on its association with corepressors or coactivators. This model would explain the apparent ability of C/EBPepsilon to both positively and negatively regulate expression of the M-CSFR gene (12, 15). Detailed characterization of the exact functions of each domain in C/EBPepsilon , and their interacting partners, along with analysis of cellular distributions of putative corepressor molecules should aid in determining the activity and specific functions of C/EBPepsilon .

    ACKNOWLEDGEMENTS

We thank Peter Johnson for advice and Curt Pfarr, Dan Hardy, Elmus Beale, Charles Faust, and Clinton MacDonald for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by grants from the National Office and Texas Affiliate of the American Heart Association, the Association for Research of Childhood Cancer, Texas Tech University Health Sciences Center, and the South Plains Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom reprint requests should be addressed: Dept. of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th St., Lubbock, TX 79430. Tel.: 806-743-2524; Fax: 806-743-2990; E-mail: cbbscw{at}ttuhsc.edu.

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; DBD, DNA binding domain; ADM, activation domain module; AD, activation domain; RD, regulatory domain; M-CSFR, macrophage-colony-stimulating factor receptor; CM, conserved motif; PCR, polymerase chain reaction; MAP, mitogen-activated protein.

2 N. D. Angerer and S. C. Williams, unpublished results.

3 A. J. Reinhart, S. C. Williams, B. J. Clark, and D. M. Stocco, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
References

  1. Shivdasani, R. A., and Orkin, S. H. (1996) Blood 87, 4025-4039[Free Full Text]
  2. Johnson, P. F., and Williams, S. C. (1994) in Liver Gene Expression (Tronche, F., and Yaniv, M., eds), pp. 231-258, R. G. Landes Co., Austin
  3. Clarke, S., and Gordon, S. (1998) J. Leukocyte Biol. 63, 153-168[Abstract]
  4. Tenen, D. G., Hromas, R., Licht, J. D., and Zhang, D.-E. (1997) Blood 90, 489-519[Free Full Text]
  5. Zhang, D.-E., Zhang, P., Wang, N., Hetherington, C. J., Darlington, G. J., and Tenen, D. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 569-574[Abstract/Free Full Text]
  6. Poli, V., and Ciliberto, G. (1994) in Liver Gene Expression (Tronche, F., and Yaniv, M., eds), pp. 131-151, R. G. Landes Co., Austin
  7. Hu, H.-M., Baer, M., Williams, S. C., Johnson, P. F., and Schwartz, R. C. (1998) J. Immunol. 160, 2334-2342[Abstract/Free Full Text]
  8. Bretz, J. D., Williams, S. C., Baer, M., Johnson, P. F., and Schwartz, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7306-7310[Abstract]
  9. Williams, S. C., Cantwell, C. A., and Johnson, P. F. (1991) Genes Dev. 5, 1553-1567[Abstract]
  10. Antonson, P., Stellan, B., Yamanaka, R., and Xanthopoulos, K. G. (1996) Genomics 35, 30-38[CrossRef][Medline] [Order article via Infotrieve]
  11. Chumakov, A. M., Grillier, I., Chumakova, E., Chih, D., Slater, J., and Koeffler, H. P. (1997) Mol. Cell. Biol. 17, 1375-1386[Abstract]
  12. Williams, S. C., Du, Y., Schwartz, R. C., Weiler, S. R., Ortiz, M., Keller, J. R., and Johnson, P. F. (1998) J. Biol. Chem. 273, 13493-13501[Abstract/Free Full Text]
  13. Morosetti, R., Park, D. J., Chumakov, A. M., Grillier, I., Shiohara, M., Gombart, A. F., Nakamaki, T., Weinberg, K., and Koeffler, H. P. (1997) Blood 90, 2591-2600[Abstract/Free Full Text]
  14. Yamanaka, R., Kim, G.-D., Radomska, H. S., Lekstrom-Himes, J., Smith, L. T., Antonson, P., Tenen, D. G., and Xanthopoulos, K. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6462-6467[Abstract/Free Full Text]
  15. Yamanaka, R., Barlow, C., Lekstrom-Hines, J., Castilla, L. H., Liu, P. P., Eckhaus, M., Decker, T., Wynshaw-Boris, A., and Xanthopoulos, K. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13187-13192[Abstract/Free Full Text]
  16. Iwama, A., Zhang, P., Darlington, G. J., McKercher, S. R., Maki, R., and Tenen, D. G. (1998) Nucleic Acids Res. 26, 3034-3043[Abstract/Free Full Text]
  17. Radomska, H. S., Huettner, C. S., Zhang, P., Cheng, T., Scadden, D. T., and Tenen, D. G. (1998) Mol. Cell. Biol. 18, 4301-4314[Abstract/Free Full Text]
  18. Williams, S. C., Baer, M., Dillner, A. J., and Johnson, P. F. (1995) EMBO J. 14, 3170-3183[Abstract]
  19. Sadowski, I., and Ptashne, M. (1989) Nucleic Acids Res. 17, 7539[Medline] [Order article via Infotrieve]
  20. Cress, W. D., and Triezenberg, S. J. (1991) Science 251, 87-90[Medline] [Order article via Infotrieve]
  21. Katz, S., Kowenz-Leutz, E., Muller, C., Meese, K., Ness, S. A., and Leutz, A. (1993) EMBO J. 12, 1321-1332[Abstract]
  22. Nalbant, D., Williams, S. C., Stocco, D. M., and Khan, S. A. (1998) Endocrinology 139, 272-279[Abstract/Free Full Text]
  23. Friedman, A. D., and McKnight, S. L. (1990) Genes Dev. 4, 1416-1426[Abstract]
  24. Pei, D. Q., and Shih, C. H. (1991) Mol. Cell. Biol. 11, 1480-1487[Medline] [Order article via Infotrieve]
  25. Nerlov, C., and Ziff, E. B. (1994) Genes Dev. 8, 350-362[Abstract]
  26. Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes Dev. 5, 1538-1552[Abstract]
  27. Williams, S. C., Angerer, N. D., and Johnson, P. F. (1997) Gene Expr. 6, 371-385[Medline] [Order article via Infotrieve]
  28. Cowell, I. G. (1994) Trends Biol. Sci. 19, 38-42[CrossRef]
  29. Fisher, A. L., and Caudy, M. (1998) Genes Dev. 12, 1931-1940[Free Full Text]
  30. Fisher, A. L., Ohsako, S., and Caudy, M. (1996) Mol. Cell. Biol. 16, 2670-2677[Abstract]
  31. Dennig, J., Beato, M., and Suske, G. (1996) EMBO J. 15, 5659-5667[Abstract]
  32. Brown, H. J., Sutherland, J. A., Cook, A., Bannister, A. J., and Kouzarides, T. (1995) EMBO J. 14, 124-131[Abstract]
  33. Karin, M. (1995) J. Biol. Chem. 270, 16483-16486[Free Full Text]
  34. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2207-2211[Abstract]
  35. Sterneck, E., and Johnson, P. F. (1998) J. Neurochem. 70, 2424-2433[Medline] [Order article via Infotrieve]
  36. Kowenz-Leutz, E., Twamley, G., Ansieau, S., and Leutz, A. (1994) Genes Dev. 8, 2781-2791[Abstract]
  37. Stein, B., Cogswell, P. C., and Baldwin, A. S., Jr. (1993) Mol. Cell. Biol. 13, 3964-3974[Abstract]
  38. Leclair, K. P., Blanar, M. A., and Sharp, P. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8145-8149[Abstract]
  39. Petrovick, M. S., Hiebert, S. W., Friedman, A. D., Hetherington, C. J., Tenen, D. G., and Zhang, D.-E. (1998) Mol. Cell. Biol. 18, 3915-3925[Abstract/Free Full Text]
  40. Nishio, Y., Isshiki, H., Kishimoto, T., and Akira, S. (1993) Mol. Cell. Biol. 13, 1854-1862[Abstract]
  41. Hsu, W., Kerppola, T. K., Chen, P.-L., Curran, T., and Chen-Kiang, S. (1994) Mol. Cell. Biol. 14, 268-276[Abstract]
  42. Lee, Y. M., Miau, L. H., Chang, C. J., and Lee, S. C. (1996) Mol. Cell. Biol. 16, 4257-4263[Abstract]
  43. Lee, Y. H., Williams, S. C., Baer, M., Sterneck, E., Gonzalez, F. J., and Johnson, P. F. (1997) Mol. Cell. Biol. 17, 2038-2047[Abstract]
  44. Chen, P. L., Riley, D. J., Chen, Y. M., and Lee, W. H. (1996) Genes Dev. 10, 2794-2804[Abstract]
  45. Chen, P.-L., Riley, D. J., Chen-Kiang, S., and Lee, W.-H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 465-469[Abstract/Free Full Text]
  46. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J. 9, 1897-1906[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.