Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide

Abstract

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. We report an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls (p = 0.0017 and 0.0006 for G/G genotype and G allele distribution, respectively), and in completed suicide cases the G(-1019) allele was enriched fourfold (p = 0.002 and 0.00008 for G/G genotype and G allele distribution, respectively). The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. Our data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. We suggest a novel transcriptional model in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide.

Figure 1.

A C(-1019)G polymorphism of the human 5-HT1A receptor promoter. A 716 bp fragment in the repressor region of the human 5-HT1A promoter was amplified by PCR and sequenced. Shown is a partial DNA sequence from PCR products of three different depressed patients that revealed the homozygous C(-1019) sequence (1), the heterozygous sequence with both C and G at the (-1019) site (2), and the homozygous G(-1019) sequence (3).

Figure 2.

Allele-specific association of a nuclear protein complex with the C(-1019) palindrome of the 5-HT1A receptor gene. EMSA was done using a 26 bp probe that includes the C(-1019)G polymorphism of the human 5-HT1A receptor gene associated with depression. Labeled 26 bp oligonucleotide probes were present in samples as the C(-1019) or the G(-1019) allele as indicated. Unlabeled 26 bp oligonucleotides (26bp-C or 26bp-G) or unrelated E2F oligonucleotides (E2F) were added at 100-fold or 1-, 2-, 3-, or 4-fold molar concentration to the incubation as indicated. A, Using RN46A nuclear extracts, a single major specific complex was observed (arrowhead) that showed preferential binding to the 26bp-C oligonucleotide. B, Decreased competition was observed with the 26bp-G oligonucleotide, which required a twofold molar excess to start competing with the 26bp-C probe for RN46A nuclear extracts. C, Nuclear extracts from rat brain tissues, including raphe-midbrain, cortex (Ctx), and hippocampus (Hip), showed specific binding to the labeled 26bp-C oligonucleotide (arrowhead). D, Yeast one-hybrid cloning of binding proteins specific for the C(-1019) palindrome allele. Six copies of the C(-1019) or G(-1019) alleles of the 26 bp element of the 5-HT1A receptor gene were integrated in the yeast genome 5′ to the LacZ gene. The two yeast strains generated were nontransformed (Ctrl) or transformed with plasmid DNA from the indicated cDNA clones (76D, 18C, or 33B). Trans-activation was measured by β-galactosidase activity in a plate assay (left) and by quantitative spectrophotometry to calculate the activity ratio of C/G (right), expressed as mean ± SD (n = 2).

Figure 3.

Trans-repression at the palindrome of the 5-HT1A receptor gene by NUDR and Hes5: differential sensitivity to the C(-1019)G polymorphism. Human 5-HT1A receptor-negative HEK 293 (A, C) or receptor-positive raphe RN46A cells (B, D, E) were cotransfected with the indicated luciferase reporter constructs and vector (pcDNA3), NUDR, or Hes5 expression plasmids, as indicated. Data are expressed as adjusted luciferase activity corrected for transfection efficiency by determining the ratio of luciferase activity/β-galactosidase activity as described (Ou et al., 2000) and are presented as mean ± SD of triplicate samples from experiments that were repeated at least twice. Statistical significance compared with vector control (for NUDR and Hes5) or promoter control (for vector) 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 < 0.05, ^**p < 0.005, ^***p < 0.0005. A, B, Repression of the human 5-HT1A promoter (-1128 bp to the initiation ATG) by NUDR and Hes5 in HEK 293 (A) and RN46A cells (B). The C(-1019) or G(-1019) allele of the -1128 bp 5-HT1A promoter-luciferase reporter construct [5-HT1A(C) and 5-HT1A(G), respectively] was cotransfected with vector, NUDR, or Hes5. Compared with vector, NUDR and Hes5 repressed 5-HT1A(C) but lacked significant activity at the G(-1019) allele, 5-HT1A(G). C, D, The 26 bp 5-HT1A palindrome mediates repression of TK promoter by NUDR and Hes5. Reporter constructs containing TK promoter alone (TK, open bars) or the 26 bp 5-HT1A palindrome C or G allele placed upstream (5′) of TK [26bp-C(TK) or 26bp-G(TK), respectively] were cotransfected with vector, NUDR, or Hes5 plasmids in HEK 293 (C) or RN46A cells (D). NUDR and Hes5 mediated repression via the 26 bp DNA elements, but only NUDR was entirely specific for the C allele. E, Repression of SV40 promoter (pGL3P, open bar) at a hexamer of 26 bp elements in RN46A cells. Six copies of the 26 bp element [26bp-C(6) or 26bp-G(6)] were placed upstream of the SV40 promoter in the pGL3P plasmid. The C-G change blocked basal repression and repression augmented by NUDR but not Hes5.

Figure 4.

Presence of NUDR bound to the C(-1019) allele in RN46A nuclear extracts. EMSA was done with the 26bp-C probe using recombinant NUDR or RN46A nuclear extracts as indicated. Unlabeled 26 bp oligonucleotides (26bp-C) or unrelated E2F oligonucleotides (E2F) were added at 100-fold molar excess to the incubation as indicated. A, In vitro-transcribed and -translated recombinant NUDR bound specifically to the 26bp-C probe. B, Binding of endogenous NUDR to the C(-1019) allele in RN46A nuclear extracts. A major specific complex was observed (left arrowhead) that was supershifted (right arrowhead) during incubation with anti-NUDR antibody; anti-NUDR alone did not bind to probe. C, Negative control experiment showing that no supershift of the protein-DNA complex in RN46A cells was induced using rabbit preimmune serum (PI). D, Localization of NUDR binding site within the proximal 5-HT1A promoter region by DNase I protection assay. A 405 bp DNA probe spanning the region between -723 and -1128 was treated with the indicated units of DNase I in the absence (a) or presence of 10 μg (b) or 20 μg (c) of raphe-midbrain nuclear extracts, or in the presence of recombinant NUDR protein (R). Midbrain extracts and NUDR protected a region centered between -1016 and -1019, corresponding to the TTCG NUDR recognition sequence and the polymorphic site [C(-1019)G]. The nucleotide position within the 5-HT1A 5′-flanking sequence is marked adjacent to the sequencing reaction (G).

Figure 5.

NUDR protein is expressed in 5-HT1A receptor-positive cells and brain regions and regulates 5-HT1A protein expression. A, B, Western blot analysis of nuclear extracts from RN46A cells (1), adult rat raphe-midbrain (2), cortex (3), and hippocampus (4) using anti-NUDR (A) or anti-Hes5 (B) antibodies. A common 33 kDa band on Coomassie-stained gel is shown as a loading control. NUDR was expressed in nuclear extracts from RN46A cells and rat brain tissues, whereas Hes5 expression was restricted to RN46A cells. C, Western blot analysis and [³H]-8-OH-DPAT binding for RN46A cells (RN) stably expressing NUDR (clones 11, B, and 15). Left panels, NUDR reduced 5-HT1A binding and 5-HT1A protein expression. β-actin immunoreactivity was tested to confirm equal loading. Right panel, Western blot analysis showing specific 5-HT1A receptor immunoreactivity in myoblast L6 cells transfected with the rat 5-HT1A expression vector but not with pcDNA3 vector, as indicated. D, Strong NUDR immunostaining was detected in the dorsal raphe nucleus (DRN) and the CA2, CA3, and dentate gyrus (DG) of the hippocampus (Hip). E, Colocalization (in yellow) of NUDR (in red) and 5-HT1A receptor or 5-HT (in green) in RN46A cells (1), primary cultures of embryonic hippocampal (2), and cortical (3) cells and dorsal raphe nucleus (4). In the dorsal raphe nuclei, some cells (indicated by an arrow) that stained for NUDR displayed weak 5-HT1A receptor or 5-HT staining.

Figure 6.

Costaining of NUDR with neuronal markers and specificity of immunohistochemical staining. A, Primary cultures of cortical (1) and hippocampal (2) cells were costained with antibodies to NUDR and neuronal markers MAP2 and TuJ1, demonstrating the presence of NUDR in neurons. B, Specificity of 5-HT, 5-HT1A, and NUDR staining in raphe nuclei. Immunofluorescence was visualized through the green and red channels as indicated using primary 5-HT1A and NUDR antibodies in the absence of secondary antibodies (primaries only). For “secondaries only,” these were no primary antibodies in the presence of both secondary antibodies. Specific immunoreactivity was observed through the green but not the red channel using anti-5-HT or anti-5-HT1A antibodies in the presence of both secondary antibodies. NUDR immunoreactivity observed through the red channel was absent in the green channel using both secondary antibodies. NUDR immunofluorescence in the presence of hNUDR (36-51) blocking peptide (100 μg) is greatly reduced.

Figure 7.

Functional model for derepression at the 5-HT1A promoter by the C(-1019)G polymorphism. The C(-1019)G polymorphism prevents binding of the transcriptional repressor NUDR resulting in enhanced 5-HT1A receptor expression in raphe neurons that may occur in development to adulthood. Increased expression of 5-HT1A somatodendritic autoreceptors would be expected to reduce serotonergic tone, which is associated with major depression. The C(-1019)G polymorphism also impairs repression by Hes5, which may play a role as a developmental regulator of 5-HT1A gene expression.