CaMKIV-Mediated Phosphorylation Inactivates Freud-1/CC2D1A Repression for Calcium-Dependent 5-HT1A Receptor Gene Induction.

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response.

Decreased expression of Freud-1/CC2D1A, a transcriptional repressor of the 5-HT1A receptor, in the prefrontal cortex of subjects with major depression.

Serotonin1A (5-HT(1A)) receptors are reported altered in the brain of subjects with major depressive disorder (MDD). Recent studies have identified transcriptional regulators of the 5-HT(1A) receptor and have documented gender-specific alterations in 5-HT(1A) transcription factor and 5-HT(1A) receptors in female MDD subjects. The 5' repressor element under dual repression binding protein-1 (Freud-1) is a calcium-regulated repressor that negatively regulates the 5-HT(1A) receptor gene. This study documented the cellular expression of Freud-1 in the human prefrontal cortex (PFC) and quantified Freud-1 protein in the PFC of MDD and control subjects as well as in the PFC of rhesus monkeys chronically treated with fluoxetine. Freud-1 immunoreactivity was present in neurons and glia and was co-localized with 5-HT(1A) receptors. Freud-1 protein level was significantly decreased in the PFC of male MDD subjects (37%, p=0.02) relative to gender-matched control subjects. Freud-1 protein was also reduced in the PFC of female MDD subjects (36%, p=0.18) but was not statistically significant. When the data was combined across genders and analysed by age, the decrease in Freud-1 protein level was greater in the younger MDD subjects (48%, p=0.01) relative to age-matched controls as opposed to older depressed subjects. Similarly, 5-HT(1A) receptor protein was significantly reduced in the PFC of the younger MDD subjects (48%, p=0.01) relative to age-matched controls. Adult male rhesus monkeys administered fluoxetine daily for 39 wk revealed no significant change in cortical Freud-1 or 5-HT(1A) receptor proteins compared to vehicle-treated control monkeys. Reduced protein expression of Freud-1 in MDD subjects may reflect dysregulation of this transcription factor, which may contribute to the altered regulation of 5-HT(1A) receptors observed in subjects with MDD. These data may also suggest that reductions in Freud-1 protein expression in the PFC may be associated with early onset of MDD.

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

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.

Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

Public perceptions of climate change as a human health risk: surveys of the United States, Canada and Malta.

We used data from nationally representative surveys conducted in the United States, Canada and Malta between 2008 and 2009 to answer three questions: Does the public believe that climate change poses human health risks, and if so, are they seen as current or future risks? Whose health does the public think will be harmed? In what specific ways does the public believe climate change will harm human health? When asked directly about the potential impacts of climate change on health and well-being, a majority of people in all three nations said that it poses significant risks; moreover, about one third of Americans, one half of Canadians, and two-thirds of Maltese said that people are already being harmed. About a third or more of people in the United States and Canada saw themselves (United States, 32%; Canada, 67%), their family (United States, 35%; Canada, 46%), and people in their community (United States, 39%; Canada, 76%) as being vulnerable to at least moderate harm from climate change. About one third of Maltese (31%) said they were most concerned about the risk to themselves and their families. Many Canadians said that the elderly (45%) and children (33%) are at heightened risk of harm, while Americans were more likely to see people in developing countries as being at risk than people in their own nation. When prompted, large numbers of Canadians and Maltese said that climate change can cause respiratory problems (78-91%), heat-related problems (75-84%), cancer (61-90%), and infectious diseases (49-62%). Canadians also named sunburn (79%) and injuries from extreme weather events (73%), and Maltese cited allergies (84%). However, climate change appears to lack salience as a health issue in all three countries: relatively few people answered open-ended questions in a manner that indicated clear top-of-mind associations between climate change and human health risks. We recommend mounting public health communication initiatives that increase the salience of the human health consequences associated with climate change.

The mental retardation gene CC2D1A/Freud-1 encodes a long isoform that binds conserved DNA elements to repress gene transcription.

The CC2D1A/Freud-1 gene has recently been linked to non-syndromic mental retardation and a short isoform of mouse Five prime REpressor Under Dual repression binding protein 1 (Freud-1) can repress the serotonin-1A (5-HT1A) receptor gene in rodent cells. In this study, we addressed the expression, localization and regulation of the human 5-HT1A receptor gene by a long isoform of human Freud-1 protein (Freud-1L). We show that human CC2D1A/Freud-1 RNA is expressed in brain and peripheral tissues and encodes short and long isoforms, which differ by an upstream in-frame translational start site. Whereas previous studies identified the short isoform of Freud-1 as the predominant isoform in rodent cells, we demonstrate that the long isoform is more abundant in human cells, especially in the nuclear fraction. The nuclear localization of Freud-1L was enriched upon inhibition of chromosome region maintenance 1/exportin 1-dependent nuclear export, indicating a dynamic regulation of Freud-1 nuclear localization. Consistent with a functional role in the nucleus, human Freud-1L bound specifically to its dual repressor element in the 5-HT1A receptor gene in vitro and repressed transcription from these sites. Importantly, chromatin immunoprecipitation using antibodies specific for human Freud-1L demonstrated that it is bound to the dual repressor element in chromatin, indicating a functional role in regulating the basal expression of the 5-HT1A receptor gene. Taken together, these results indicate that both the short and long isoforms of Freud-1 are expressed, although Freud-1L is the major isoform that regulates the human 5-HT1A receptor gene. Disruption of transcriptional regulation by mutation of Freud-1 may play a role in abnormal brain function leading to mental retardation.

The Freud-1/CC2D1A family: transcriptional regulators implicated in mental retardation.

The CC2D1A gene family consists of two homologous genes, Freud-1/CC2D1A and Freud-2/CC2D1B, that share conserved domains, including several DM14 domains that are specific to this protein family, a C-terminal helix-loop-helix domain, and a C2 calcium-dependent phospholipid binding domain. Although the function of Freud-2 is unknown, Freud-1 has been shown to function as a transcriptional repressor of the serotonin-1A receptor gene that binds to a novel DNA element (FRE, 5'-repressor element). The DNA binding and repressor activities of Freud-1 are inhibited by calcium-calmodulin-dependent protein kinase. Recently, a deletion in the CC2D1A gene has been linked to nonsyndromic mental retardation. This deletion results in the truncation of the helix-loop-helix DNA binding and the C2 domains, crucial for Freud-1 repressor activity, and hence is predicted to generate an inactive or weakly dominant negative protein. The possible mechanisms by which inactivation of Freud-1 could lead to abnormal cortical development and cognitive impairment and the potential roles of Freud-1 gene targets are discussed.

Differential repression by freud-1/CC2D1A at a polymorphic site in the dopamine-D2 receptor gene.

Freud-1/CC2D1A is a transcriptional repressor of the serotonin-1A receptor gene and was recently genetically linked to non-syndromic mental retardation. To identify new Freud-1 gene targets, data base mining for Freud-1 recognition sequences was done. A highly homologous intronic element (D2-DRE) was identified in the human dopamine-D2 receptor (DRD2) gene, and the role of Freud-1 in regulating the gene at this site was assessed. Recombinant Freud-1 bound specifically to the D2-DRE, and a major protein-D2-DRE complex was identified in nuclear extracts that was supershifted using Freud-1-specific antibodies. Endogenous Freud-1 binding to the D2-DRE in cells was detected using chromatin immunoprecipitation. The D2-DRE conferred strong repressor activity in transcriptional reporter assays that was dependent on the Freud-1 recognition sequence. In three different human cell lines, the level of Freud-1 protein was inversely related to DRD2 expression. Knockdown of endogenous Freud-1 using small interfering RNA resulted in an up-regulation of DRD2 RNA and binding sites, demonstrating a crucial role for Freud-1 in DRD2 regulation. A previously uncharacterized single nucleotide A/G polymorphism (rs2734836) was located adjacent to the D2-DRE and conferred allele-specific Freud-1 binding and repression, with the major G-allele having reduced activity. These studies demonstrate a key role for Freud-1 to regulate DRD2 expression and provide the first mechanistic insights into its transcriptional regulation. Allele-specific regulation of DRD2 expression by Freud-1 may possibly associate with psychiatric disorders or mental retardation.

Cell type-dependent recruitment of trichostatin A-sensitive repression of the human 5-HT1A receptor gene.

Regulation of serotonin (5-HT)1A receptor expression in brain is implicated in mood disorders such as depression and anxiety. Transcriptional activity of the human 5-HT1A receptor gene was strongly repressed by a negative regulatory region containing a consensus repressor element-1 (RE-1) and two copies of the dual repressor element (DRE) identified in the rat 5-HT1A receptor gene. REST/NRSF, a silencer of neuronal genes, bound the 5-HT1A RE-1 and repressed the 5-HT1A promoter. Inactivation of RE-1 completely abolished REST-mediated repression, but resulted in only partial (15-50%) de-repression of basal 5-HT1A promoter activity. The human 5-HT1A DRE sequences bound specifically to the novel repressor Freud-1 (5'repressor element under dual repression binding protein-1) and conferred repressor activity at 5-HT1A or SV40 promoters. In 5-HT1A-negative cells [L6, human embryonic kidney (HEK) 293], the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) abolished repression mediated by both RE-1/REST and DRE/Freud-1, and induced almost complete de-repression of the 5-HT1A gene. By contrast, in 5-HT1A-expressing neuronal cells (RN46A, SN-48) TSA blocked RE-1/REST repression, but did not affect DRE/Freud-1-mediated repression. Thus in contrast to REST, Freud-1 mediates HDAC-independent repression of the 5-HT1A receptor promoter in neuronal 5-HT1A-positive cells, suggesting that HDAC recruitment might influence neuron-specific gene expression by further silencing expression in non-neuronal tissue.