Melanocortin-4 Receptor Promoter Research Articles

Regulation of melanocortin-4-receptor (MC4R) expression by SNP rs17066842 is dependent on glucose concentration

Melanocortin-4-receptor (MC4R) gene codes for a G-protein-coupled receptor that is highly expressed in the hypothalamus and involved in the regulation of appetite. Single-nucleotide polymorphisms (SNPs) in the MC4R gene region have been associated with obesity, type 2-diabetes (T2D) and with antipsychotic-induced weight gain. Of these, rs17066842 (G>A) in the MC4R promoter region is the top variant associated with obesity and diabetes. In this study, we investigated the effect of rs17066842 on MC4R expression at various glucose concentrations using reporter gene expression in the SH-SY5Y cell line and regulation of MC4R expression in human cerebral organoids. We observed that higher glucose concentrations significantly reduced MC4R mRNA expression in SH-SY5Y cells. In addition, at high glucose concentrations, the luciferase reporter plasmid containing the MC4R promoter insert with the G-allele of rs170066842 showed significantly reduced activity compared to the A-allele carrying plasmid. The immediate early gene product, early growth-response 1 (EGR-1), was identified to bind to the sequence containing the G-allele at rs17066842 but not to the A-allele-containing sequence. Interestingly, in human induced pluripotent stem cell (hiPSC)-derived cerebral organoids, we observed increased MC4R expression in response to high glucose exposure. These opposite observations might suggest that glucose regulation is complex and may be cell-specific.This study provides evidence that rs17066842 regulates MC4R gene expression through binding of EGR-1 and that this process is influenced by glucose concentration.

MC4R and HNF4α promoter methylation at birth contribute to triglyceride levels in childhood: A prospective cohort study.

Although the changes in DNA methylation are assumed to be due to the association between adverse intrauterine conditions and adult metabolic health, evidence from human studies is rare. Little is known about the changes in DNA methylation present at birth that affect metabolic profiles in childhood. Previous studies have shown that the melanocortin 4 receptor (MC4R) and hepatocyte nuclear factor 4 alpha (HNF4α) genes are associated with obesity and metabolic disorders. Thus, we investigated the associations of the DNA methylation statuses of MC4R and HNF4α in cord blood with metabolic profiles in childhood.We collected data from 90 children 7 to 9 years of age included in the Ewha Birth & Growth Cohort Study in Korea. DNA methylation was analyzed by pyrosequencing. The children were split into 2 groups according to the cutoff triglyceride (TG) levels (<110 and ≥110 mg/dL).The methylation statuses of MC4R and HNF4α at birth were significantly associated with the TG level in childhood (P < .05). It was interesting to note that the methylation statuses of MC4R and HNF4α in cord blood were significantly decreased, whereas childhood body mass index was significantly increased, in children with high TG levels compared with children with low TG levels (P < .05).Our findings show that the methylation statuses of MC4R and HNF4α at birth are associated with metabolic profiles in childhood. These epigenetic modifications occurring in early life may contribute to subsequent metabolic-related disorders. Thus, we suggest that DNA methylation status in cord blood may be predictive of the risk of developing metabolic syndrome.

Variation in the Promoter Region of the MC4R Gene Elucidates the Association of Body Measurement Traits in Hu Sheep.

The melanocortin 4 receptor (MC4R) gene is expressed in the appetite-regulating areas of the brain and is engaged in the leptin signaling pathway. Although previous studies have identified variants in the coding region of the sheep MC4R gene showing significant associations with birth weight, weaning weight, and backfat thickness, no such associations have been reported for the promoter region. Besides, the essential promoter region of the sheep MC4R has not been delineated. In this study, to better understand the transcriptional regulation of MC4R and to elucidate the association between regulatory variants and haplotypes with body measurement traits in sheep, we cloned and characterized the MC4R promoter. We found that the minimal promoter of the gene is located within the region −1207/−880 bp upstream of the first exon. Real-time quantitative PCR (RT-qPCR) data revealed the mRNA expression of the MC4R gene had a significant difference between sex and age. In the association analysis, eight single nucleotide polymorphisms (SNPs) had a significant association with one or more traits (p < 0.05); of these, two SNPs were novel. Notably, individuals with haplotype H1H2 (CT-GA-GT-GA-GT-GA-GA-CG) were heavier in body weight than other haplotypes. Altogether, variations in the MC4R gene promoter, most notably haplotype H1H2, may greatly benefit marker-assisted selection in sheep.

Melanocortin-4 receptor expression in the rostral ventromedial medulla involved in modulation of nociception in transgenic mice

The rostral ventromedial medulla (RVM) is a prominent component of the descending modulatory system involved in the control of spinal nociceptive transmission. In the current study, we investigated melanocortin-4 receptor (MC4R) expression in the RVM, where the neurons involved in modulation of nociception reside. Using a line of mice expressing green fluorescent protein (GFP) under the control of the MC4R promoter, we found a large number of GFP-positive neurons in the RVM [nucleus raphe magnus (NRM) and nucleus gigantocellularis pars α (NGCα)]. Fluorescence immunohistochemistry revealed that approximately 10% of MC4R-GFP-positive neurons coexpressed tyrosine hydroxylase, indicating that they were catecholaminergic, whereas 50%-75% of those coexpressed tryptophan hydroxylase, indicating that they were serotonergic. Our findings support the hypothesis that MC4R signaling in RVM may modulate the activity of serotonergic sympathetic outflow sensitive to nociceptive signals, and that MC4R signaling in RVM may contribute to the descending modulation of nociceptive transmission.

HSF is the Most Important Transcriptional Factor for Porcine MC4R Promoter

The melanocortin-4 receptor (MC4R) is involved in feed intake regulation. It is significantly associated with growth and fatness traits in most breeds and crosses. To understand the essential transcriptional regions of the porcine MC4R promoter, eleven primer pairs were designed to amplify different segment lengths of the MC4R promoter. The PCR products were then ligated along with the Firefly luciferase reporter gene into the PGL3-basic vector. The results showed that a 90 bp fragment could contain the essential regions for control of transcription. Further research found that a short sequence “AGAAAGAAG” (the recognition site of transcription factor HSF) was the most important sequence for supporting activity of the pig MC4R promoter.

Melanocortin-4 receptor expression in a vago-vagal circuitry involved in postprandial functions.

Vagal afferents regulate energy balance by providing a link between the brain and postprandial signals originating from the gut. In the current study, we investigated melanocortin-4 receptor (MC4R) expression in the nodose ganglion, where the cell bodies of vagal sensory afferents reside. By using a line of mice expressing green fluorescent protein (GFP) under the control of the MC4R promoter, we found GFP expression in approximately one-third of nodose ganglion neurons. By using immunohistochemistry combined with in situ hybridization, we also demonstrated that approximately 20% of GFP-positive neurons coexpressed cholecystokinin receptor A. In addition, we found that the GFP is transported to peripheral tissues by both vagal sensory afferents and motor efferents, which allowed us to assess the sites innervated by MC4R-GFP neurons. GFP-positive efferents that co-expressed choline acetyltransferase specifically terminated in the hepatic artery and the myenteric plexus of the stomach and duodenum. In contrast, GFP-positive afferents that did not express cholinergic or sympathetic markers terminated in the submucosal plexus and mucosa of the duodenum. Retrograde tracing experiments confirmed the innervation of the duodenum by GFP-positive neurons located in the nodose ganglion. Our findings support the hypothesis that MC4R signaling in vagal afferents may modulate the activity of fibers sensitive to satiety signals such as cholecystokinin, and that MC4R signaling in vagal efferents may contribute to the control of the liver and gastrointestinal tract.

Melanocortin‐4 receptor expression in a vago‐vagal circuitry involved in postprandial functions

AbstractVagal afferents regulate energy balance by providing a link between the brain and postprandial signals originating from the gut. In the current study, we investigated melanocortin‐4 receptor (MC4R) expression in the nodose ganglion, where the cell bodies of vagal sensory afferents reside. By using a line of mice expressing green fluorescent protein (GFP) under the control of the MC4R promoter, we found GFP expression in approximately one‐third of nodose ganglion neurons. By using immunohistochemistry combined with in situ hybridization, we also demonstrated that ∼20% of GFP‐positive neurons coexpressed cholecystokinin receptor A. In addition, we found that the GFP is transported to peripheral tissues by both vagal sensory afferents and motor efferents, which allowed us to assess the sites innervated by MC4R‐GFP neurons. GFP‐positive efferents that co‐expressed choline acetyltransferase specifically terminated in the hepatic artery and the myenteric plexus of the stomach and duodenum. In contrast, GFP‐positive afferents that did not express cholinergic or sympathetic markers terminated in the submucosal plexus and mucosa of the duodenum. Retrograde tracing experiments confirmed the innervation of the duodenum by GFP‐positive neurons located in the nodose ganglion. Our findings support the hypothesis that MC4R signaling in vagal afferents may modulate the activity of fibers sensitive to satiety signals such as cholecystokinin, and that MC4R signaling in vagal efferents may contribute to the control of the liver and gastrointestinal tract. J. Comp. Neurol. 518:6–24, 2010. © 2009 Wiley‐Liss, Inc.

Analysis of Single Nucleotide Polymorphisms in the Melanocortin-4 Receptor Promoter in Infantile Spasms

Infantile spasms is a severe epileptic encephalopathy of infancy. ACTH that ameliorates infantile spasms might act through activating the central melanocortin-4 receptor (MC4R) to suppress excessive production of corticotrophin-releasing hormone (CRH). This study aimed to elucidate an association between the genetic variants of the MC4R gene and infantile spasms. The study population comprised 96 patients with infantile spasms and 118 controls. All subjects were screened for variations in the promoter and coding region of the MC4R gene using a direct sequencing method. ATCH responses in patients carrying different genotypes were also assessed. The distributions of genotypes and alleles of rs11872992 in the MC4R promoter were significantly different between cases and controls. The frequencies of heterozygous carriers (TC genotype) were significantly lower in cases (10%) than in controls (27%) (p=0.003). The distributions of rs11872992 TC and CC genotypes were significantly different between ACTH responders and non-responders (OR, 0.14; 95% C.I, 0.03-0.69; p=0.007). The T-allele carriers (83.3%) had a higher responsiveness to ACTH than non-carriers (41.7%). The present study shows that genetic variants in the MC4R promoter are associated with the development of infantile spasms. The rs11872992 polymorphism influences ACTH treatment responses in patients with infantile spasms.

A Two-Base Deletion –439delGC in the Melanocortin-4 Receptor Promoter Associated with Early-Onset Obesity

Background/Aims: Mutations in melanocortin-4 receptor (MC4R) are the most common genetic cause of human obesity. Mutations in MC4R promoter could also underlie obesity, but have so far not been reported. Transcription factor nescient helix-loop-helix 2 (Nhlh2) is a novel obesity candidate gene. We searched for mutations in MC4R promoter and Nhlh2 gene in 152 children with severe early-onset obesity. Lean subjects (n = 447) served as controls. Methods: MC4R promoter and Nhlh2 gene were investigated by sequencing. Gel shifts and reporter gene assays were used to investigate a deletion in MC4R promoter. Mutation carriers were carefully characterised. Weight charts from index patients and relatives were analysed. Results: We identified a deletion, –439delGC, in MC4R promoter in 2 severely obese, unrelated children and their family members, but not in controls. Index patients and mutation-carrying relatives were affected by early-onset obesity, while non-carriers had normal childhood weight development. The deletion is located at a potential Nhlh2-binding site and gel shift assays showed that Nhlh2 binds to this site. No significant differences in mutant compared to wild-type MC4R promoter activities were detected. No mutations were identified in Nhlh2 gene. Conclusion: We report an MC4R promoter mutation, –439delGC, associated with early-onset obesity and show that transcription factor Nhlh2 recognises this site in vitro. Nhlh2 mutations unlikely underlie severe human obesity.

1 kb of 5′ flanking sequence from mouse MC4R gene is sufficient for tissue specific expression in a transgenic mouse

The melanocortin 4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis, and the MC4R knockout mouse and humans with MC4R defective mutations in only one allele indicate that there is a gene dosage effect. Alterations in gene expression levels for MC4R could, therefore, have significant effects on energy homeostasis. To begin to develop a mouse model for studies on MC4R promoter in situ we used ∼1 kb mouse MC4R promoter together with 426 bp MC4R 5′ UTR, previously shown to support basal expression of reporter gene transcription in cell lines with endogenous MC4R mRNA, and fused this DNA to a nuclear localized LacZ reporter gene. The construct was injected into pronuclei from FVB mice. Five transgenic lines were identified as carrying autosomal transgene insertions; three of these had significant β-galactosidase staining in brain and in a few cells in the heart but not in kidney, liver, lung, gonadal fat or testis. The pattern of transgene expression in the brain differed markedly for the three lines, and in one of these lines was remarkably similar to endogenous MC4R mRNA expression observed using in situ hybridisation. In conclusion, ∼1 kb mouse MC4R promoter is sufficient to direct gene expression to the brain including regions that express endogenous MC4R mRNA.

The human MC4R promoter: characterization and role in obesity.

Heterozygous mutations in the coding sequence of the serpentine melanocortin 4 receptor (MC4R) are the most frequent genetic cause of severe human obesity. Since haploinsufficiency has been proposed as a causal mechanism of obesity associated with these mutations, reduction in gene transcription caused by mutations in the transcriptionally essential regions of the MC4R promoter may also be a cause of severe obesity in humans. To test this hypothesis we defined the minimal promoter region of the human MC4R and evaluated the extent of genetic variation in this region compared with the coding region in two cohorts of severely obese subjects. 5'RACE followed by functional promoter analysis in multiple cell lines indicates that an 80-bp region is essential for the transcriptional activity of the MC4R promoter. Systematic screening of 431 obese children and adults for mutations in the coding sequence and the minimal core promoter of MC4R reveals that genetic variation in the transcriptionally essential region of the MC4R promoter is not a significant cause of severe obesity in humans.

Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Melanocortin-4 Receptor Promoter

The melanocortin-4 receptor (MC4-R) is an important regulator of energy homeostasis, and evidence suggests that MC4-R-expressing neurons are downstream targets of leptin action. MC4-Rs are broadly expressed in the CNS, and the distribution of MC4-R mRNA has been analyzed most extensively in the rat. However, relatively little is known concerning chemical profiles of MC4-R-expressing neurons. The extent to which central melanocortins act presynaptically or postsynaptically on MC4-Rs is also unknown. To address these issues, we have generated a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the MC4-R promoter, using a modified bacterial artificial chromosome. We have confirmed that the CNS distribution of GFP-producing cells is identical to that of MC4-R mRNA in wild-type mice and that nearly all GFP-producing cells coexpress MC4-R mRNA. For example, cells coexpressing GFP and MC4-R mRNA were distributed in the paraventricular hypothalamic nucleus (PVH) and the dorsal motor nucleus of the vagus (DMV). MC4-R promotor-driven GFP expression was found in PVH cells producing thyrotropin-releasing hormone and in cholinergic DMV cells. Finally, we have observed that a synthetic MC3/4-R agonist, MT-II, depolarizes some GFP-expressing cells, suggesting that MC4-Rs function postsynaptically in some instances and may function presynaptically in others. These studies extend our knowledge of the distribution and function of the MC4-R. The transgenic mouse line should be useful for future studies on the role of melanocortin signaling in regulating feeding behavior and autonomic homeostasis.

Putative targets of CNS melanocortin receptor activity.

Chronic antagonism of hypothalamic melanocortin receptors, primarily melanocortin-4 receptor (MC4R), is the molecular basis for "agouti obesity syndrome," whereas suppression of MC4R gene activity due to genetic mutations induces obesity in both rodents and humans. However, little is known about the neurocircuitry of MC4R-mediated control of energy balance, the regulation of MC4R gene expression, or how suppression of MC4R activity leads to differential expression of potential downstream central nervous system (CNS) targets or effectors of melanocortin signaling. This paper focuses on strategies for mapping CNS melanocortin circuits using transgenic mouse models for conditional expression of MC4R and MC3R as well as progress in characterizing the murine MC4R promoter. Additionally, preliminary studies that focus on putative targets of melanocortinergic signaling will include a discussion of CD81, a gene identified using the polymerase chain reaction-based method of suppression subtractive hybridization. CD81, first described as TAPA-1 (target of antiproliferative antibody), is a member of the tetraspanin family of cell surface proteins believed to function in cell-cell adhesion, signal transduction, and possibly neuronal plasticity. Elevated expression of CD81 mRNA in hypothalamic regions of obese yellow mice suggests that loss of MC4R activity may lead to altered neuronal function via modulation of the cell surface protein CD81.