Cell-specific repressor or enhancer activities of Deaf-1 at a serotonin 1A receptor gene polymorphism

Abstract

The serotonin-1A (5-HT1A) receptor is the primary somatodendritic autoreceptor that inhibits the activity of serotonergic raphe neurons and is also expressed in nonserotonergic cortical and limbic neurons. Alterations in 5-HT1A receptor levels are implicated in mood disorders, and a functional C(-1019)G 5-HT1A promoter polymorphism has been associated with depression, suicide, and panic disorder. We examined the cell-specific activity of identified transcription factors, human nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR)/Deaf-1 and Hes5, at the 5-HT1A C(-1019) site. In serotonergic raphe RN46A cells, Deaf-1 and Hes5 repressed the 5-HT1A receptor gene at the C(-1019)-allele but not the G(-1019)-allele. However, in nonserotonergic cells that express 5-HT1A receptors (septal SN48, neuroblastoma SKN-SH, and neuroblastoma/glioma NG108-15 cells), Deaf-1 enhanced 5-HT1A promoter activity at the C(-1019)-allele but not the G-allele, whereas Hes5 repressed in all cell types. The enhancer activity of Deaf-1 was orientation independent and competed out Hes5 repression. To test whether Deaf-1 activity is intrinsic, the activity of a Gal4DBD (DNA binding domain)-Deaf-1 fusion protein at a heterologous Gal4 DNA element was examined. Gal4DBD-Deaf-1 repressed transcription in RN46A cells but enhanced transcription in SN48 cells, indicating that these opposite activities are intrinsic to Deaf-1. Repressor or enhancer activities of Deaf-1 or Gal4DBD-Deaf-1 were blocked by histone deacetylase inhibitor trichostatin A. Thus, the intrinsic activity of Deaf-1 at the 5-HT1A promoter is opposite in presynaptic versus postsynaptic neuronal cells and requires deacetylation. Cell-specific regulation by Deaf-1 could underlie region-specific alterations in 5-HT1A receptor expression in different mood disorders.

Figure 1.

Cell type-dependent activity of Deaf-1 at the 5-HT_1A C(-1019) site. Constructs containing the C(-1019) or G(-1019) allele of the human 5-HT_1A promoter (−1128 bp to ATG) fused to luciferase [5-HT_1A(C) and 5-HT_1A(G), respectively] were cotransfected with vector (pcDNA3), Deaf-1, or Hes5 expression plasmids as indicated in 5-HT_1A-expressing neuronal cell lines: rat raphe RN46A (A), mouse septal SN-48, neuroblastoma-glioma NG108–15, or human SKN-SH neuroblastoma cells (B). Adjusted luciferase activity was corrected for transfection efficiency by calculating the ratio of luciferase activity/β-galactosidase activity and normalized to control (pcDNA3) transfections. Data are presented as mean ± SD of triplicate samples collected from at least three independent experiments as indicated. Statistical analysis compared with vector control (for Deaf-1 and Hes5) was determined by one-way ANOVA; significant differences between C and G alleles were evaluated by unpaired t test with two-tailed p values (**p ≤ 0.01; ***p ≤ 0.001).

Figure 2.

Endogenous Deaf-1 protein expression and DNA binding activity. A, Deaf-1 localization. Nuclear (N) and cytosolic (C) extracts (50 μg/lane) from RN46A, SN48, SKN-SH, and NG108–15 cells were subjected to Western blot analysis using specific anti-Deaf-1 antibody. The blot was reprobed using antibodies for markers of nuclear (histone-H1) and cytosolic (c-Raf) fractions to assess loading and purity of extracts. Molecular weight markers (kDa) are as indicated. B, Deaf-1 DNA binding activity. Electrophoretic mobility shift assay was done using nuclear extracts from RN46A, SN48, or NG108–15 cells that were incubated with labeled 26-bp(C) probe in the presence of 100-fold molar excess of unlabeled 26 bp(C) (Sp) or unrelated PEA3 (NSp) oligonucleotides, as indicated. A major specific Deaf-1 complex was observed (arrow).

Figure 3.

Orientation-independent repressor and enhancer activities of Deaf-1 at the 5-HT_1A palindrome. In RN46A (A) or SN48 (B) cells, luciferase reporter constructs containing six copies of the 26 bp 5-HT_1A palindrome in the forward [26bp-C(6)F] or reverse orientation [26bp-C(6)R] relative to the SV40 promoter were cotransfected with vector (pcDNA3) or Deaf-1 expression plasmid. Luciferase activity was normalized to vector control, and data are presented as mean ± SD of triplicate samples collected from four independent experiments. Statistical analysis was performed by unpaired t test with two-tailed p values (***p ≤ 0.001).

Figure 4.

Deaf-1 competes with Hes5 to enhance 5-HT_1A promoter activity. The 26bp-C(6) reporter construct [containing 6 copies of the C(-1019) allele of the 26 bp 5-HT_1A palindrome] or the 5-HT_1A(C) promoter construct was cotransfected with vector (pcDNA3), Deaf-1, or Hes5 plasmids in RN46A cells (A) or SN48 cells (B, C). Luciferase activity was normalized to control transfections. Data are presented as mean ± SEM of triplicate samples from three separate experiments. Significance is compared with vector control as indicated (**p < 0.01; ***p < 0.001).

Figure 5.

Repressor and enhancer activities of Deaf-1 at the 5-HT_1A promoter are TSA sensitive. The −1128 bp 5-HT_1A-luciferase construct [5-HT_1A(C)] was cotransfected with vector (pcDNA3) or Deaf-1 plasmids in rat raphe RN46A and mouse septal SN-48 cells. As a positive control for TSA, a luciferase construct containing human 5-HT_1A RE-1 placed 5′ to SV40-promoter was cotransfected with vector (pcDNA1) or REST plasmids. Cells were treated with vehicle or TSA (200 nm) for 24 h, as indicated. Luciferase activity (Luc. Act.)/β-galactosidase activity was normalized to control (vector) transfections. Data are presented as mean ± SD of triplicate samples collected from at least three independent experiments, as indicated. Statistical analysis was determined by unpaired t test with two-tailed p values (***p ≤ 0.001).

Figure 6.

Cell-specific and TSA-sensitive repressor and enhancer activities are conferred by Deaf-1 at a heterologous DNA element. A, A model for the action of effectors Gal4-DBD (Gal4 vector), Gal4DBD-Deaf-1 fusion protein, or LexA (positive control) at the X2G2P reporter construct containing two LexA and Gal4 sites upstream of SV40 promoter-luciferase. These constructs were cotransfected in 5-HT_1A-expressing rat RN46A (B) or mouse SN48 (C) cells, which were treated for 24 h with vehicle or TSA (200 nm). Luciferase activity was normalized to Gal4 vector (100%). Data are presented as mean ± SD of triplicate samples collected from at least two independent experiments as indicated. Statistical analysis was determined by unpaired t test with two-tailed p values (***p ≤ 0.001).