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Functional genomics of serotonin receptor 2A (HTR2A): interaction of polymorphism, methylation, expression and disease association.

Falkenberg VR, Gurbaxani BM, Unger ER, Rajeevan MS - Neuromolecular Med. (2010)

Bottom Line: This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner.This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches.Confirmation in an independent study population is required.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral and Rickettsial Diseases, Centers for Disease Control & Prevention, Atlanta, GA 30333, USA.

ABSTRACT
Serotonergic neurotransmission plays a key role in the pathophysiology of neuropsychiatric illnesses. The functional significance of a promoter polymorphism, -1438G/A (rs6311), in one of the major genes of this system (serotonin receptor 2A, HTR2A) remains poorly understood in the context of epigenetic factors, transcription factors and endocrine influences. We used functional and structural equation modeling (SEM) approaches to assess the contributions of the polymorphism (rs6311), DNA methylation and clinical variables to HTR2A expression in chronic fatigue syndrome (CFS) subjects from a population-based study. HTR2A was up-regulated in CFS through allele-specific expression modulated by transcription factors at critical sites in its promoter: an E47 binding site at position -1,438, (created by the A-allele of rs6311 polymorphism), a glucocorticoid receptor (GR) binding site encompassing a CpG at position -1,420, and Sp1 binding at CpG methylation site -1,224. Methylation at -1,420 was strongly correlated with methylation at -1,439, a CpG site that is dependent upon the G-allele of rs6311 at position -1,438. SEM revealed a strong negative interaction between E47 and GR binding (in conjunction with cortisol level) on HTR2A expression. This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner. This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches. Confirmation in an independent study population is required.

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Analyses of GR binding and a qualitative regulatory model of HTR2A expression integrating genetic, epigenetic and disease associations. The 5′end of the HTR2A promoter is shown at the top with relevant experimentally verified TFBS defined. The putative + or − effect on transcription when bound and the location of CpGs in the sequence are also shown. Methylation sites are indicated by  when largely unmethylated and by  when largely methylated. Loss of methylation site due to polymorphism is indicated by . The effect of genotype and methylation on TF binding, and thus HTR2A transcription is indicated by the size of the arrow at the end of each model. The model predicts subjects with AA genotype largely explain the differential expression of HTR2A between CFS and NF subjects. Inhibition of E47 binding by cortisol-bound GR in NF subjects is indicated by ⊣ and the inhibited transcription factor is covered by
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Fig5: Analyses of GR binding and a qualitative regulatory model of HTR2A expression integrating genetic, epigenetic and disease associations. The 5′end of the HTR2A promoter is shown at the top with relevant experimentally verified TFBS defined. The putative + or − effect on transcription when bound and the location of CpGs in the sequence are also shown. Methylation sites are indicated by when largely unmethylated and by when largely methylated. Loss of methylation site due to polymorphism is indicated by . The effect of genotype and methylation on TF binding, and thus HTR2A transcription is indicated by the size of the arrow at the end of each model. The model predicts subjects with AA genotype largely explain the differential expression of HTR2A between CFS and NF subjects. Inhibition of E47 binding by cortisol-bound GR in NF subjects is indicated by ⊣ and the inhibited transcription factor is covered by

Mentions: Multiple lines of evidence identified transcription factors that regulate HTR2A expression through binding at positions −1,438, −1,420 and −1,224; E47 binds at −1,439, glucocorticoid receptor (GR) binds at −1,420 and Sp1 binds at −1,224 (Fig. 1; Supplementary Table 1). Binding of Sp1 at −1,224 was identified as early as 1995 through sequencing, gel retardation and DNase I footprinting assays (Zhu et al. 1995) and also conserved in Rattus norvegicus and Mus musculus. Recently, we demonstrated multiple lines of evidence for the binding of GR at −1,420 through computational analysis, competitive electrophoretic mobility shift assays (EMSA) using both HeLA and purified GR, and showing that this GRE binding site is highly conserved (79–95%) in Rattus norvegicus, Mus musculus and Pan troglodytes (Falkenberg and Rajeevan 2010). Experimental verification of E47 binding at −1,438 specific to the A-allele of HTR2A was reported recently by our group using computational and competitive EMSA (Smith et al. 2008). We also found this E47 binding site to be conserved in Mus musculus (76%). Based on these substantial computational and experimental evidences for these different binding sites, we propose a qualitative regulatory model of HTR2A expression (Fig. 5). In this model, increased methylation of the CpG at position −1,224 in NF subjects would result in reduced binding of the Sp1 transcriptional activator and an overall lower expression of HTR2A. Higher HTR2A expression in CFS subjects, particularly higher expression of the A allele, would be accounted for by the creation of a binding site for the transcriptional activator E47 at −1,438, along with the deficiency of GR binding at −1,420 under a state of hypocortisolism (Heim et al. 2000) and the potential for increased methylation at this site.Fig. 5


Functional genomics of serotonin receptor 2A (HTR2A): interaction of polymorphism, methylation, expression and disease association.

Falkenberg VR, Gurbaxani BM, Unger ER, Rajeevan MS - Neuromolecular Med. (2010)

Analyses of GR binding and a qualitative regulatory model of HTR2A expression integrating genetic, epigenetic and disease associations. The 5′end of the HTR2A promoter is shown at the top with relevant experimentally verified TFBS defined. The putative + or − effect on transcription when bound and the location of CpGs in the sequence are also shown. Methylation sites are indicated by  when largely unmethylated and by  when largely methylated. Loss of methylation site due to polymorphism is indicated by . The effect of genotype and methylation on TF binding, and thus HTR2A transcription is indicated by the size of the arrow at the end of each model. The model predicts subjects with AA genotype largely explain the differential expression of HTR2A between CFS and NF subjects. Inhibition of E47 binding by cortisol-bound GR in NF subjects is indicated by ⊣ and the inhibited transcription factor is covered by
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044825&req=5

Fig5: Analyses of GR binding and a qualitative regulatory model of HTR2A expression integrating genetic, epigenetic and disease associations. The 5′end of the HTR2A promoter is shown at the top with relevant experimentally verified TFBS defined. The putative + or − effect on transcription when bound and the location of CpGs in the sequence are also shown. Methylation sites are indicated by when largely unmethylated and by when largely methylated. Loss of methylation site due to polymorphism is indicated by . The effect of genotype and methylation on TF binding, and thus HTR2A transcription is indicated by the size of the arrow at the end of each model. The model predicts subjects with AA genotype largely explain the differential expression of HTR2A between CFS and NF subjects. Inhibition of E47 binding by cortisol-bound GR in NF subjects is indicated by ⊣ and the inhibited transcription factor is covered by
Mentions: Multiple lines of evidence identified transcription factors that regulate HTR2A expression through binding at positions −1,438, −1,420 and −1,224; E47 binds at −1,439, glucocorticoid receptor (GR) binds at −1,420 and Sp1 binds at −1,224 (Fig. 1; Supplementary Table 1). Binding of Sp1 at −1,224 was identified as early as 1995 through sequencing, gel retardation and DNase I footprinting assays (Zhu et al. 1995) and also conserved in Rattus norvegicus and Mus musculus. Recently, we demonstrated multiple lines of evidence for the binding of GR at −1,420 through computational analysis, competitive electrophoretic mobility shift assays (EMSA) using both HeLA and purified GR, and showing that this GRE binding site is highly conserved (79–95%) in Rattus norvegicus, Mus musculus and Pan troglodytes (Falkenberg and Rajeevan 2010). Experimental verification of E47 binding at −1,438 specific to the A-allele of HTR2A was reported recently by our group using computational and competitive EMSA (Smith et al. 2008). We also found this E47 binding site to be conserved in Mus musculus (76%). Based on these substantial computational and experimental evidences for these different binding sites, we propose a qualitative regulatory model of HTR2A expression (Fig. 5). In this model, increased methylation of the CpG at position −1,224 in NF subjects would result in reduced binding of the Sp1 transcriptional activator and an overall lower expression of HTR2A. Higher HTR2A expression in CFS subjects, particularly higher expression of the A allele, would be accounted for by the creation of a binding site for the transcriptional activator E47 at −1,438, along with the deficiency of GR binding at −1,420 under a state of hypocortisolism (Heim et al. 2000) and the potential for increased methylation at this site.Fig. 5

Bottom Line: This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner.This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches.Confirmation in an independent study population is required.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral and Rickettsial Diseases, Centers for Disease Control & Prevention, Atlanta, GA 30333, USA.

ABSTRACT
Serotonergic neurotransmission plays a key role in the pathophysiology of neuropsychiatric illnesses. The functional significance of a promoter polymorphism, -1438G/A (rs6311), in one of the major genes of this system (serotonin receptor 2A, HTR2A) remains poorly understood in the context of epigenetic factors, transcription factors and endocrine influences. We used functional and structural equation modeling (SEM) approaches to assess the contributions of the polymorphism (rs6311), DNA methylation and clinical variables to HTR2A expression in chronic fatigue syndrome (CFS) subjects from a population-based study. HTR2A was up-regulated in CFS through allele-specific expression modulated by transcription factors at critical sites in its promoter: an E47 binding site at position -1,438, (created by the A-allele of rs6311 polymorphism), a glucocorticoid receptor (GR) binding site encompassing a CpG at position -1,420, and Sp1 binding at CpG methylation site -1,224. Methylation at -1,420 was strongly correlated with methylation at -1,439, a CpG site that is dependent upon the G-allele of rs6311 at position -1,438. SEM revealed a strong negative interaction between E47 and GR binding (in conjunction with cortisol level) on HTR2A expression. This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner. This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches. Confirmation in an independent study population is required.

Show MeSH
Related in: MedlinePlus