Transcription factor GATA-3 regulates the transcriptional activity of dopamine beta-hydroxylase by interacting with Sp1 and AP4

Abstract

GATA-3 is a zinc finger transcription factor that is expressed in T cell lineages as well as in the nervous system during development. In this study, we report that forced expression of GATA-3 resulted in an increased number of dopamine beta-hydroxylase (DBH)-expressing neurons in primary neural crest stem cell (NCSC) culture, suggesting that the DBH gene may be a downstream target gene of GATA-3. GATA-3 robustly transactivates the promoter function of the noradrenaline (NA)-synthesizing DBH gene, via two specific upstream promoter domains; one at -62 to -32 bp and the other at -891 to -853 bp. Surprisingly, none of these domains contain GATA-3 binding sites but encompass binding motifs for transcription factors Sp1 and AP4, respectively. Protein-protein interaction analyses both in vitro and in vivo and chromatin immunoprecipitation (ChIP) assays showed that GATA-3 effects its transcriptional regulatory function through physical interactions with these transcription factors.

Fig. 1

GATA-3 overexpression increases the number of DBH expressing cells in neural crest cell culture. Primary neural crest cell cultures were infected with RCASBP(B) alone or RCAS-cGATA3 viruses. ISH were performed to detect DBH expression. DBH positive cells are represented as a percentage. Data are presented as mean ±SEM from four independent experiments. Bar: 300 μm.

Fig. 2

GATA-3 regulates expression of DBH. (a) The expression of DBH is reduced by GATA-3 siRNA. SK-N-BE(2)C cells were transfected with GATA-3 siRNA (lane 2, 4) or control siRNA (lane 1, 3) using Lipofectamine. After 24 (lane 1, 2) or 72 h (lane 3, 4) of transfection, cells were harvested and Western blot analyses were performed. The expression of GATA-3, DBH, Sp1, AP4 was detected with their specific antibodies as indicated. β-actin was detected as a loading control. (b) GATA-3 transactivation of serially deleted DBH promoter. HeLa cells were cotransfected with DBH-CAT reporter plasmids and pcDNA/GATA3 or empty vector at a molar ratio of 0.2. Fold induction by effector plasmid cotransfection is presented as mean ± SEM value from six to nine independent samples. The numbers on the left of the diagram represent the size of the DBH promoter. The bent arrow represents the DBH transcription start site. The bold thick line denotes the 5′ untranslated sequences and the thin line denotes the 5′ upstream sequences of the DBH gene. The arrowhead represents the GATA-3 response region of the DBH promoter.

Fig. 3

GATA-3 physically interacts with Sp1. (a) Mutation of Sp1 binding site in DBH978CAT diminishes reporter gene activation by GATA-3. The bold characters represent the Sp1 binding site. (b) Localization of the interaction domains of GATA-3 and Sp1. In vitro translated [³⁵S]-methionine labeled GATA-3 proteins were incubated with GST (lanes 6, 8, 10, 12, and 14) or with full-length GST-Sp1 (lanes 7, 9, 11, 13, and 15) bound to glutathione Sepharose beads. [³⁵S]-methionine labeled input proteins are shown (lanes 1 (aa 1-144), 2 (aa 1-364), 3 (aa 1-248), 4 (aa 242-364) and 5 (aa 268-364)). The numbers at the top of the figure represent the amino acid residues of GATA3. (c) Interaction between GATA-3 and Sp1 in vivo. 293T cells were cotransfected with the Sp1 (aa 613 - 716), and empty vector or GATA-3 as indicated on the top. The cell lysates were precipitated with glutathione-Sepharose beads. Monoclonal anti-FLAG was used to detect the FLAG-tagged GATA-3 proteins. The blot was stripped and re-probed with the anti-GST to detect the precipitated Sp1proteins (bottom). Top, crude cell extracts; middle, immunoprecipitates; bottom, GST-Sp1. Asterisk indicates non-specific bands containing the heavy chain of IgG. (d) Summary of the interaction results of GATA3 with Sp1. Three zinc finger motifs (Zf1-3) of Sp1 are indicated.

Fig. 4

GATA-3 transactivates the DBH promoter through AP4 binding site. (a) GATA-3 transactivation analysis of serially deleted human DBH promoter. (b) The AP4 binding site (CAGCTG) was mutated. Cotransfections were performed in HeLa cells and the same symbols are used as in Fig. 2.

Fig. 5

AP4 binds to the E-box motif of the DBH upstream promoter. Oligonucleotide DBH/AP4 containing the E-box (CANNTG) motif of the DBH promoter was radiolabeled and used as a probe. (a) Two μl of the in vitro translated AP4 proteins were incubated with the radiolabeled probe (lane 2) and 0.2 μg of AP4 antibody was added to the reaction (lane 3). For competition, 50-fold (lanes 4, 7, 10), 200-fold (lanes 5, 8, 11), or 1,000-fold (lanes 6, 9, 12) molar excess of unlabeled oligonucleotides DBH/AP4 (lanes 4-6), Sp1 (lanes 7-9), and SV40/AP4 (10-12) were added to the reaction mixture before the addition of radiolabeled probe. The same amount of in vitro translated protein using an empty vector was incubated with the probe and did not generate any complex (lane 1). (b, c) The ³²P-labeled DBH/AP4 probe was incubated with 3 μg of HeLa (b) or 1 μg of SK-N-BE(2)C (c) nuclear extracts (lane 2, 9). For competition, 50-fold (lanes 3, 6), 200-fold (lanes 4, 7), or 1,000-fold (lanes 5, 8) molar excess of unlabeled oligonucleotides DBH/AP4 (lanes 3-5) and Sp1 (lanes 6-8) were added to the reaction mixture before the addition of radiolabeled probe. AP4 specific antibody was incubated with the probe and nuclear extracts (lane 10). In lane 1, the probe was incubated without nuclear extracts. (d) The ³²P-labeled DBH/AP4 probe was incubated with 2 μl of in vitro translated AP4 proteins (lane 2). For competition, 50-fold (lanes 3, 6), 200-fold (lanes 4, 7), or 1,000-fold (lanes 5, 8) molar excess of unlabeled oligonucleotide wild type DBH/AP4 (lanes 3-5) and mutant type DBH/AP4 (lanes 6-8) were used. The same amounts of in vitro translated proteins using an empty vector were incubated with the probe and did not generate any complex (lane 1). Specific protein-DNA complex and supershifted complex were indicated by an arrow head and an arrow, respectively.

Fig. 6

GATA-3 physically interacts with AP4. (a, b) Determination of the interaction domains of GATA-3 and AP4. (a) In vitro translated [³⁵S]-methionine labeled GATA-3 proteins were incubated with GST (lanes 4, 6, and 8) or full length GST-AP4 (lanes 5, 7, and 9), bound to glutathione Sepharose beads. [³⁵S]-methionine labeled input proteins are shown (lanes 1 (aa 1-144), 2 (aa 1-364), and 3 (aa 242-444)). (b) In vitro translated [³⁵S]-methionine labeled AP4 proteins were incubated with GST (lanes 5, 7, 9, and 11) or full-length GST-GATA3 (lanes 6, 8, 10, and 12) bound to glutathione Sepharose beads. [³⁵S]-methionine labeled input proteins are shown (lanes 1 (aa 1-338), 2 (aa 1-253), 3 (aa 1-182), and 4 (aa 1-98)). (c) Interaction between GATA-3 and AP4 in vivo. 293T cells were cotransfected with the empty vector, GATA-3, and the AP4 as indicated on the top. The cell lysates were precipitated with an anti-GFP. Monoclonal anti-FLAG was used to detect the FLAG-tagged AP4 proteins. The blot was stripped and re-probed with the anti-GFP to detect the precipitated GATA-3 protein (bottom). Top, crude cell extracts; middle, immunoprecipitates; bottom, GFP-GATA3. Asterisk indicates non-specific bands containing the heavy chain of IgG. (d) Schematic representation of interactions between GATA-3 and AP4. The basic (B) region, helix-loop-helix (HLH) domain, and two leucine zipper domains (LZ1, 2) are indicated.

Fig. 7

ChIP analysis indicates in vivo interaction of GATA-3 with SP1 and AP4 on the DBH promoter. (a) Schematic drawing of primers to detect Sp1 and AP4 binding sites by PCR. The first exon of human DBH gene is shown (Exon I). The numbers indicate nucleotide position in the DBH promoter. (b) The protein-DNA complexes were immunoprecipitated using antibodies against GATA-3 (lane 3, 6). As a negative control, rabbit IgG was used (lane 2, 5). Lane 1 and 4 show input DNAs. PCR was performed with primer sets shown (a) as described in experimental procedure. One twelfth (lanes 1, 4) or one third (lanes 2, 3, 5, 6) of the reaction PCR products were run in 7% polyacrylamide gel.