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Published on: May 13, 2019
Polymorphism in Tmem132d regulates expression and anxiety-related behavior through binding of RNA polymerase II
Roshan R Naik1,2,3, Sergey V Sotnikov4,5, Rebekka P Diepold4
1Max Planck Institute of Psychiatry, 80804, Munich, Germany. rosnaik@gmail.com.
Abstract:
TMEM132D is a candidate gene, where risk genotypes have been associated with anxiety severity along with higher mRNA expression in the frontal cortex of panic disorder patients. Concurrently, in a high (HAB) and low (LAB) trait anxiety mouse model, Tmem132d was found to show increased expression in the anterior cingulate cortex (aCC) of HAB as compared to LAB mice. To understand the molecular underpinnings underlying the differential expression, we sequenced the gene and found two single-nucleotide polymorphisms (SNPs) in the promoter differing between both lines which could explain the observed mRNA expression profiles using gene reporter assays. In addition, there was no difference in basal DNA methylation in the CpG Island that encompasses the HAB vs. LAB Tmem132d promoter region. Furthermore, we found significantly higher binding of RNA polymerase II (POLR2A) to the proximal HAB-specific SNP (rs233264624) than the corresponding LAB locus in an oligonucleotide pull-down assay, suggesting increased transcription. Virus mediated overexpression of Tmem132d in the aCC of C57BL/6 J mice could confirm its role in mediating an anxiogenic phenotype. To model gene-environmental interactions, HAB mice exposed to enriched environment (HAB-EE) responded with decreased anxiety levels but, had enhanced Tmem132d mRNA expression as compared to standard-housed HAB (HAB-SH) mice. While LAB mice subjected to unpredictable chronic mild stress (LAB-UCMS) exhibited higher anxiety levels and had lower mRNA expression compared to standard-housed LAB (LAB-SH) mice. Chromatin immunoprecipitation revealed significantly higher binding of POLR2A to rs233264624 in HAB-EE, while LAB-UCMS had lower POLR2A binding at this locus, thus explaining the enhanced or attenuated expression of Tmem132d compared to their respective SH controls. To further investigate gene-environment interactions, DNA methylation was assessed using Illumina 450 K BeadChip in 74 panic disorder patients. Significant methylation differences were observed in two CpGs (cg26322591 and cg03283235) located in TMEM132D depending on the number of positive life events supporting the results of an influence of positive environmental cues on regulation of Tmem132d expression in mice.
Insights
TMEM132D gene expression influences anxiety severity. Genetic variations and environmental factors modulate its expression, impacting anxiety phenotypes in both mice and humans.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- TMEM132D is a candidate gene linked to anxiety disorders.
- Higher TMEM132D mRNA expression in the frontal cortex is observed in panic disorder patients.
- Differential Tmem132d expression exists between high (HAB) and low (LAB) trait anxiety mouse models in the anterior cingulate cortex (aCC).
Purpose of the Study:
- To investigate the molecular mechanisms underlying differential Tmem132d expression in anxiety.
- To explore the role of genetic variations and gene-environment interactions in Tmem132d regulation.
- To determine the functional impact of Tmem132d on anxiety-related behaviors.
Main Methods:
- Gene sequencing and reporter assays to identify functional genetic variations.
- Oligonucleotide pull-down and chromatin immunoprecipitation assays to assess protein binding and transcription.
- Virus-mediated gene manipulation in mice to study Tmem132d function.
- Environmental manipulations (enriched environment, chronic mild stress) and DNA methylation analysis in mice and human patients.
Main Results:
- Two promoter single-nucleotide polymorphisms (SNPs) in Tmem132d were identified between HAB and LAB mice, correlating with mRNA expression.
- Increased RNA polymerase II (POLR2A) binding at the HAB-specific SNP (rs233264624) suggests higher transcription.
- Overexpression of Tmem132d in mice induced an anxiogenic phenotype.
- Environmental enrichment (EE) in HAB mice decreased anxiety but increased Tmem132d expression, while stress in LAB mice increased anxiety and decreased expression, linked to POLR2A binding.
- Human panic disorder patients showed methylation differences in TMEM132D CpG sites correlating with life events.
Conclusions:
- TMEM132D promoter SNPs and differential POLR2A binding contribute to anxiety-related gene expression.
- Tmem132d plays a causal role in mediating anxiety-like behaviors.
- Gene-environment interactions significantly influence TMEM132D expression and anxiety levels, with epigenetic modifications potentially playing a role.
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