Binding of transcription factors to specific sequences is important for post-transcriptional regulation. Various studies have shown that 5' regulatory region polymorphisms are closely associated with changes in gene expression. For example, rs2239630G/A is present in the promoter of the CCAT enhancer-binding protein epsilon (CEBPE) gene. The rs2239630A allele can enhance promoter activity and CEBPE expression and is associated with the risk of acute lymphoblastic leukemia. The Matrix metalloproteinase (MMP-9) gene promoter polymorphism − 1562C/T regulates its transcriptional activity and affects the course of thyroid carcinoma.
In this study, six haplotype recombinants were transfected into SK-N-SH and HEK-293. The H2 and H4 haplotypes were associated with decreased transcriptional activity, whereas haplotype H6 increased transcriptional activity in both cell lines. In our previous study, we observed that the distribution of haplotype H6 was different between schizophrenic patients and healthy individuals in the northern Han Chinese population; however, the statistical difference was lost after performing the Bonferroni correction. Schizophrenia is a complex polygenetic disease. Haplotype C-G-C-A-G-A (H6), which is found in rs4140535, rs1778258, rs17273700, rs1228814, rs11568817, and rs130058, significantly increased HTR1B gene expression levels, which could have potential effects in schizophrenia. It should be noted that haplotype H2 caused the lowest HTR1B expression in both SK-N-SH and HEK-293. Although there was no evidence that this haplotype is associated with schizophrenia based on our previous studies, we cannot rule out that it may be related to other mental disorders caused by 5-HT1B dysfunction.
We also focused on the effects of HTR1B SNPs on gene expression. We found that the rs4140535T allele could enhance transcriptional activity. Previous studies found that 5-HT1B plays an important role in regulating feeding behavior. HTR1B polymorphism G861C was associated with minimum lifetime body mass indices (BMIs) in female patients with bulimia nervosa. Another study showed that rs4140535 could be a genetic factor for obesity in African Americans, and the rs4140535T allele was a protective allele against excessive BMI. Blocking 5-HT1B eliminates the anorexia and weight loss caused by leptin and AM 251, and serotonin-induced decreases in appetite require activation of 5-HT1B . Our data suggest that the HTR1B polymorphism rs4140535T/C is likely related to weight gain. Our results also show that the rs1778258A allele has a negative regulatory effect on HTR1B gene expression. In our previous study, we found that the rs1778258A allele was potentially associated with schizophrenia in the northern Han Chinese population and speculated that this result might be related to the effect of the rs1778258A/G polymorphism on gene expression. The function of rs1778258A/G was confirmed in the current study.
We found that the C-G combination in rs17273700 and rs11568817 upregulated HTR1B expression, and rs17273700 and rs11568817 had a strong linkage relationship. Duan et al. confirmed that the rs11568817G allele could generate a new TFAP2 binding site, resulting in improved transcriptional activity. The TFAP2 family, which includes TFAP2A, TFAP2B, and TFAP2C, represents a group of transcriptional activators that regulate the expression of specific genes. rs11568817 is also believed to be related to other psychiatric disorders, such as drug abuse, alcohol and nicotine dependence, and anxiety. It is likely related to the sexual dimorphism of the serotoninergic system. However, the function of rs17273700 needs further exploration. The rs1228814A allele could enhance transcriptional activity based on the results. Although there have been studies on the relationship between rs1228814 and depression and methamphetamine addiction[30, 31], there is currently no evidence associating rs1228814 with mental illness. Our study also found that the rs130058T allele could significantly downregulate gene expression. While rs130058 has been confirmed to bind to TFAP1, the rs130058A and rs130058T alleles exhibit different characteristics when binding to TFAP1, which affects the transcriptional activity of HTR1B.
Not all of the specific binding sequences for transcription factors included polymorphisms. Ten different truncated fragments were transfected into SK-N-SH and HEK-293 cells. The trends of the changes in relative fluorescence intensity between D0 and D1, D2 and D3, D6 andD7, and D7 and D8 were upward, while the trends for changes between D4 and D5 and D8 and D9 were downward in both cell lines. Therefore, the deleted fragments ranging from − 1587 to -1371 bp (D0-D1), -1149 to -894 bp (D2-D3), -39 ~ + 130 bp (D6 ~ D7), and + 130 ~ + 341 bp (D7 ~ D8) contain transcriptional suppression regions, and − 603~-316 bp (D4-D5), + 341 ~ + 505 bp (D8-D9) contain enhancing regulatory regions for gene expression. The functional regions were analyzed by JASPAR (http://jaspar.genereg.net), and we found many transcription factors that probably bind to HTR1B, such as TBX2, E2F6, TFAP2, and KLF3 (Fig. 4).
The bioinformatics platform predicted that the deleted fragment from − 1587 to -1371 bp (D0-D1) contains the binding site for T-box transcription factor 2 (TBX2). This transcription factor is a transcriptional repressor of ADAM10. Its effects are mediated by binding to two TBX2 binding sites within the core promoter region of ADAM10, and substrate cleavage by ADAM10 has been implicated in pathological situations such as Alzheimer's disease.. We also found that E2F6 binding sites were present in the deleted fragments from − 1149 to -894 bp (D2-D3) and − 39 to + 130 bp (D6-D7). E2F6 plays a vital role in controlling the cell cycle and contains a modular suppression domain that has inhibitory effects on transcription[33, 34]. Our analysis also predicted that the deleted fragment from + 130 to + 341 bp (D7-D8) contains a binding site for Kruppel-like factor 3 (KLF3). KLF3 belongs to the family of Sp1/Kruppel-like zinc finger transcription factors. These transcription regulators modulate the expression of a large number of genes that have GC-rich promoters and may take part in all facets of cellular function. KLF3 is widely expressed as a transcriptional repressor that functions by binding with the co-repressor protein C-terminal binding protein (CtBP).
Deletion of the fragment from − 603 to -316 bp (D4-D5) caused a decrease in transcription activity. This region contained a binding site for TFAP2C. Studies have shown that TFAP2C plays a role in cerebral cortex development, it directly regulates the basal progenitor fate determinants Tbr2 and NeuroD.. TFAP2C can also control hippocampal neurogenesis in adults and regulate cognitive behavior. It was impressive that the luciferase activity of D8 was 7 to 8-fold higher than that of D9 in SK-N-SH cells. Therefore, the truncated fragment + 341 ~ + 505 bp (D8-D9) is likely to be the core promoter region of HTR1B and plays a decisive role in its transcription.
This study investigated the function of the 5' regulatory region of HTR1B. However, the mechanisms of the functional polymorphisms still require further exploration, and the potential functional regions must be verified with additional experiments. We attempt to perform animal experiments in the following studies to investigate the regulatory mechanism of the downstream pathways and on the correlation between the function of HTR1B and psychiatric disorders.