RESULTS

Characterization of the transcriptional start sites of APOBEC3G by 5′-RACE
To identify the transcriptional start sites (TSS) of APOBEC3G (A3G) in A3.01 T cells, we performed 5′-rapid amplification of cDNA ends analysis (RACE) with A3G-specific primers (see Figure 1). Agarose gel electrophoresis resolved the nested PCR products into three bands of different electrophoretic mobility with a dominant middle band (Figure 2). For each band, the DNA was cloned and sequence analysis of six or seven individual transformants was performed. We observed that the transcriptional start sites of the A3G gene were located between 58 and 361 nt upstream of the ATG start codon (Figure 1). Although TSS were variable and most sites were only detected once among the 19 clones analyzed, one TSS was identified in six individual clones. This TSS was located 66 nt upstream of the start of the published A3G mRNA sequence (GenBank™ accession number NM021822) and we defined this position as the major transcriptional start site of the A3G gene.
Figure 1. Sequence of the A3G promoter and the downstream region. The first 1000 bp upstream of the major TSS are shown in lower case, the first 800 bp of the transcribed sequence are shown in upper case. Introns are removed, but their positions are indicated. Arrows refer to transcriptional start sites and their observed frequency is given by the numbers above. The primer binding sites for 5′-RACE analysis and cloning of the luciferase reporter constructs are underlined and the names of the primers are annotated. Gray and black arrowheads define the regions designated E1 and E2 which were cloned into vector pGL3-Promoter and used as EMSA probes. The ATG start codon and the identified Sp1/Sp3 transcription factor binding site are shown in bold.
Figure 2. 5′-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5′-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.

The core promoter of A3G is located within the region −114/+66 relative to the TSS
For characterization of the A3G promoter, we cloned the 1025 bp located at position −959/+66 relative to the identified transcription start into the promoterless pGL3-Basic luciferase reporter plasmid and designated the plasmid pGL3-Basic-APOprom1025. Similarly, pGL3-APOprom502 (containing sequence −436/+66), pGL3-APOprom225 (containing sequence −159/+66) and further 5′ deletion reporter constructs containing 180, 150, 120 or 60 bp upstream of position +65 were generated (Figure 3A). In order to analyze transcriptional activity, the luciferase reporter plasmids were transiently transfected into A3.01 T cells. Luciferase assays revealed a ∼20-fold increased transcriptional activity of the 1025 bp sequence as compared to the empty vector, indicating that we had identified an active A3G promoter sequence (Figure 3B). This transcription rate was not significantly altered by the 5′ deletions leading to the 502, 225 and 180 bp fragments (Figure 3B). In contrast, a drop in luciferase activity was observed in the case of the 150 bp fragment. This construct only retained 28% of the transcriptional activity of the 180 bp promoter in A3.01 T cells and activity of the 120 bp fragment was further reduced. Comparable reductions relative to the activity of 180 bp fragment were observed in the myeloid cell line U937 and the hepatic cell lines HepG2 and Huh7 (Figure 3C), indicating that the core promoter of A3G is located within the region −114/+66 relative to the TSS.
Figure 3. Luciferase activities of A3G promoter constructs in different cell lines. (A) A3G promoter 5′ deletion constructs of different sizes were cloned into pGL3-Basic luciferase reporter plasmids. Numbering is relative to the major TSS. A putative Sp1/Sp3 consensus site (gray square) is depicted. (B) A3.01 T cells were transiently transfected with the A3G promoter deletion constructs. Numbers on the x-axis refer to the length of the A3G promoter fragments in bp. (C) A3G promoter plasmids were transfected into U937, HepG2 and Huh7 cell lines. Numbers in the legends refer to the length of the A3G promoter fragments in bp. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown.

The A3G promoter is not inducible in T cells
According to the current knowledge, APOBEC3G plays a role in the innate defence against pathogens like HIV-1. The latter has evolved a mechanism to counteract A3G activity by expressing the regulatory protein Vif (4). Vif induces proteasomal degradation of A3G and additionally inhibits A3G activity by further mechanisms (8, 37–43). To investigate whether the overexpression of HIV-1 proteins also influences A3G promoter activity, we either expressed HIV-1 Vif or the HIV-1 Tat protein, the latter has been shown to activate different viral and cellular promoters (44,45). In addition, increasing amounts of the plasmid pNL4-3, containing the full-length genome of HIV-1, were transfected. These constructs or the empty vector pcDNA3.1 were cotransfected with the 1025 bp construct into A3.01 T cells. Luciferase assays showed that the transcriptional activity of the A3G promoter was not significantly altered in the presence of Vif, Tat or HIV-1NL4-3 (Figure 4A), suggesting that HIV-1 is not modulating A3G expression on the transcriptional level.
Figure 4. A3G promoter activities after coexpression of HIV-1 proteins or treatment with TPA or interferons. (A) A3.01 T cells were cotransfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter and 1 µg of Vif expression plasmid, 1 µg Tat expression plasmid or increasing amounts of HIV-1NL4-3 (0.1, 0.5 and 1 µg). After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (B) A3.01 T cells were transiently transfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter or with empty vector. Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 20 ng/ml TPA. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities and the values for the empty vectors (untreated and TPA-stimulated) were set as 1. (C) A3.01 T cells were transiently transfected with the A3G promoter constructs or with the interferon-responsive reporter plasmid pGL2-CVX (GAS). Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 30 ng/ml IFN-α or IFN-γ. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (D) HepG2 cells were used for transfection. The experiment was performed as described in (C). Mean values (±SD) of representative experiments performed in triplicate are shown.
It has been shown that the amount of A3G mRNA in T cells is increased in response to mitogenic stimulation with phorbol ester (46–48). In addition, interferons have been described to upregulate A3G expression in hepatocytes and macrophages (46,48). To investigate whether these stimuli interfere with A3G promoter activity in T cells, we transfected A3.01 T cells with the 1025 bp promoter or the empty vector pGL3-Basic (vector) and treated the cells with phorbol ester (TPA). The luciferase assay showed that the ∼15-fold increased transcriptional activity of the 1025 bp promoter relative to the empty vector was not further enhanced by TPA treatment (Figure 4B), although the functional activity of TPA was confirmed by induction of the SV40 promoter-containing reporter plasmid pGL3-Control (data not shown). Similarly, treatment of A3.01 T cells transfected with the A3G promoter deletion constructs with IFN-α or IFN-γ showed no effect (Figure 4C). Interestingly, a control plasmid (pGL2-CVX) containing two IFN-responsive GAS (gamma activated sequence) elements upstream of the luciferase reporter gene was only induced by IFN-α in these cells (Figure 4C). Since two reports describe A3G upregulation by interferons in hepatocytes (47,48), we additionally performed the experiment in the hepatic cell line HepG2. In line with these publications, we observed an induction of the A3G promoter by approximately 2-fold after IFN-α or IFN-γ stimulation (Figure 4D) with IFN-γ being slightly more potent. For both interferon types, induction of A3G promoter activity was observed for all deletion constructs except for the 60 bp fragment, indicating that the responsible region is located within the 60 nt present in the 120 bp, but not in the 60 bp fragment.

A GC-box located at position −87/−78 of the A3G promoter is important for transcriptional activity
The reporter studies shown in Figure 3 had demonstrated a drop in luciferase activity after deletion of the 30 nt at the 5′ end of the 180 bp core promoter. We therefore inspected the 30 bp sequence deleted in the 150 bp fragment and identified a GC-box at position −87/−78 (see Figure 1). The sequence TGGGCGGGAC, which is interrupted in the 150 bp fragment, represents a variant of the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif recognized by Sp1 and Sp3 transcription factors. To analyze whether this putative Sp1/Sp3-binding site mediates transcriptional activity of the 180 bp core promoter, we introduced two point mutations which changed the sequence from TGGGCGGGAC to TGTTCGGGAC (mutations shown in bold). This resulted in a 71% reduction of the transcriptional activity compared to the unmodified 180 bp promoter (Figure 5A) and this value was only marginally higher than the luciferase activity of the 150 bp fragment, indicating that the identified motif is essential for basal activity of the A3G core promoter. To further examine the transcriptional potency of the 30 nt present in the 180 bp promoter, we cloned the region −114/−85 (containing all nucleotides which are deleted in the 150 bp fragment, designated E1) or the region −92/−63 (containing the putative Sp1/Sp3 motif, designated E2) into the vector pGL3-Promoter (see Figure 1). This vector contains a luciferase reporter gene under the control of an SV40 promoter without enhancer sequences, and putative transcriptionally active sequences can be cloned upstream of the SV40 promoter. Luciferase assays showed that the E1 element increased SV40 promoter activity only by ∼2-fold (Figure 5B). In contrast, the 30 nt of E2 enhanced the transcriptional activity of the SV40 promoter by ∼4.3-fold, indicating that the intact GC-box present in the E2 element was responsible for the strongly enhanced transcriptional activity of the SV40 promoter.
Figure 5. A GC-box mediates transcriptional activity of the 180 bp core promoter. (A) A3.01 T cells were transfected with reporter plasmid pGL3-Basic containing 180, 150 or 120 bp of the A3G promoter. The two G-to-T substitutions introduced into the GC-box of the 180 bp fragment (180mut) are specified. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown. (B) pGL3-Promoter reporter plasmids containing the regions E1 or E2 (see Figure 1) upstream of the SV40 promoter were transfected into A3.01 T cells. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown.

Sp1 and Sp3 transcription factors bind to the GC-box at position −87/−78 of the A3G promoter
To investigate whether the GC-box represents a binding site for the transcription factors Sp1 and Sp3, we performed EMSA analyses with nuclear extracts isolated from A3.01 T cells. As probes, we radioactively labeled the unmodified E2 sequence (nucleotides −92/−63 of the A3G promoter, probe designated APO-Sp1/3) or the same region carrying the two point mutations described above (probe designated APO-Sp1/3mut). As control, we used a commercially available Sp1 consensus oligonucleotide (Sp1cons), which is known to be recognized by Sp1 transcription factors. Four DNA–protein complexes were observed in the presence of the APO-Sp1/3 probe (Figure 6A). The upper two complexes were specific since they disappeared in the presence of a 30-fold molar excess of unlabeled APO-Sp1/3 probe (Figure 6A, lane 7). Further confirmation of the specificity of these DNA–protein complexes was demonstrated by the inability of the unlabeled APO-Sp1/3mut probe to abolish binding (Figure 6A, lanes 12 and 13). As expected, the two specific complexes were also present in the case of the Sp1cons control probe (Figure 6A, lanes 2 and 3). In contrast, none of these specific complexes was observed when the mutated probe was used (Figure 6A, lanes 10 and 11), confirming that protein binding was dependent on the identified GC-box. In order to characterize the complexes, we performed supershift experiments using Sp1- and Sp3-specific antibodies. The upper of the complexes that appeared in combination with the Sp1cons or APO-Sp1/3 probes, shifted in the presence of the Sp1 antibody (Figure 6B, lanes 3 and 7), whereas the lower complex shifted in the presence of the Sp3 antibody (Figure 6B, lanes 4 and 8). Taken together, the EMSA demonstrated that the GC-box located on the A3G core promoter serves as a binding site for Sp1 and Sp3.
Figure 6. Sp1 and Sp3 bind to the GC-box present in the A3G promoter. (A) Nuclear extracts of A3.01 T cells were incubated with a 32P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein–DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). (B) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody–protein–oligo complexes (lanes 3, 4, 7, 8, 11, 12). (C) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.
To show binding of Sp1 and Sp3 factors also in the context of the endogenous A3G promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Sp1 and Sp3 antibodies, but not an actin antibody, immunoprecipitated the A3G promoter in A3.01 T cells (Figure 6C, upper panel). In contrast, no PCR signal was received with a primer pair recognizing a region in the A3G gene ∼4000 bp downstream of the Sp1/Sp3–binding site (Figure 6C, lower panel). Only the positive controls showed a DNA band, with an A3G expression plasmid or the sheared and cross-linked input DNA used as template. This demonstrates the binding of Sp1 and Sp3 transcription factors to the endogenous A3G promoter.

Silencing of Sp1 and Sp3 reduces A3G promoter activity
To confirm the role of Sp1 and Sp3 in regulation of A3G promoter activity, we silenced their translation via RNA interference. Functionality of the siRNAs directed against Sp1 or Sp3 was confirmed by western blot analysis: protein levels of Sp1 as well as the long and short isoforms of Sp3 were strongly reduced in the presence of 150 or 300 ng specific siRNA (Figure 7A). An unspecific control siRNA had no influence (Figure 7A). We then cotransfected the luciferase reporter plasmid containing the 180 bp A3G promoter together with 100 ng siRNA using an optimized protocol for the cotransfection of plasmid plus siRNA. This resulted in a 31–43% reduction of luciferase activity in the presence of Sp1- or Sp3-specific siRNA compared to the control siRNA. In contrast, no influence on transcriptional activity of the 150 bp fragment, which does not contain the Sp1/Sp3-binding motif, was observed. Thus, both Sp1 and Sp3 factors are mediating transcriptional activity of the A3G promoter.
Figure 7. Silencing of Sp1 and Sp3 reduces A3G promoter activity. (A) HeLa cells were transfected with 150 and 300 ng of unspecific, Sp1-specific or Sp3-specific siRNA. After 48 h, cells were harvested and Sp1 and Sp3 proteins were detected by western blot analysis. As loading control, protein levels of tubulin are shown. (B) HeLa cells were cotransfected with reporter plasmid pGL3-Basic containing 180 or 150 bp of the A3G promoter and siRNA. Hundred nanogram of unspecific siRNA (control), Sp1-specific siRNA (Sp1), Sp3-specific siRNA (Sp3) or a mixture of 50 ng Sp1-specific plus 50 ng Sp3-specific siRNA (Sp1+Sp3) were used. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown.