Elucidation of molecular mechanisms at the jObes1 locus causing the juvenile obesity in the Berlin fat mouse

Die Berlin Fett Maus Inzuchtlinie (BFMI) ist ein Modell für jugendliche Fettleibigkeit, die natürliche Mutationen aufweist, welche uns helfen können, die genetischen Mechanismen zu verstehen, die zu Fettleibigkeit führen. Bei früheren Untersuchungen, von Kreuzungen zwischen BFMI und B6N, wurde ein rezessiver Defekt auf Chromosom 3 (jObes1) identifiziert, der juvenile Fettleibigkeit verursacht. Die Feinkartierung engte den jObes1-Lokus, mit Hilfe einer fortgeschrittenen Kreuzungslinie auf das Bardet-Biedl-Syndrom 7 - Gen (Bbs7), das wahrscheinlichste Kandidatengen für den Phänotyp der juvenilen Fettleibigkeit in BFMI ein. Die Genexpressionsanalyse des gesamten Gehirns von BFMI- und B6N-Mäusen zeigte, dass die Expression des Gens Bbs7 bei BFMI-Mäusen im Vergleich zu den B6N-Referenzmäusen deutlich reduziert war. Sequenzvergleiche zwischen den beiden Linien BFMI und B6N zeigten zwei wesentliche Unterschiede zwischen ihnen: (1) eine 1.578 bp Deletion im Intron 8 von Bbs7 in BFMI-Mäusen, die einen CCCTC-bindenden Faktor (CTCF-Element) enthält (2) 16 Sequenzvarianten, die in der Bbs7-Promotorregion (36. 613.319 - 36.614.267, Ensembl release 102) in den BFMI-Mäusen, im Vergleich zur B6N-DNA-Sequenz, für die Unterschiede in der Bbs7-Genregulation verantwortlich sein könnten. Mit dieser Studie sollten zwei Hauptfragen beantwortet werden: (1) Hat die gelöschte Intron-8-Region von Bbs7, die ein CTCF-Element enthält, einen teilweisen oder vollständigen Einfluss auf die Entwicklung von juveniler Adipositas bei BFMI-Mäusen? (2) Was ist die ursächliche Variante in der Promotorregion des Bbs7-Gens, die zu Expressionsunterschieden des Bbs7-Gens zwischen BFMI und B6N führt? Sowohl der einzelne 5'-UTR-SNP, als auch die Deletion im Intron 8 von Bbs7, können zu dem beobachteten Phänotyp der BFMI-Mäuse beitragen, indem sie höchstwahrscheinlich die Bbs7-Expression verringern und den Fettanteil erhöhen.The Berlin Fat Mouse Inbred (BFMI) line is a model for juvenile obesity which harbors natural mutations that can help us understand the genetic mechanisms leading to obesity. Previous research on crosses between BFMI and B6N identified a recessive defect causing juvenile obesity on chromosome 3 (jObes1). This explains around 40% of the bodyweight differences in an F2 cross between BFMI and the reference B6N mouse. Fine mapping, using an advanced intercross line, of the jObes1 locus, revealed the Bardet-Biedl syndrome 7 (Bbs7) gene as the most likely candidate gene for the juvenile obesity phenotype in the BFMI. Gene expression analysis on the whole brain of BFMI and B6N mice showed that the expression of the Bbs7 gene in BFMI mice was reduced significantly compared to the B6N reference mice. Sequence comparisons between the two lines BFMI and B6N showed two major differences between them: (1) a 1,578 bp deletion in intron 8 of Bbs7 in BFMI mice harboring a CCCTC-binding factor (CTCF-element) which works as a transcriptional activator, a repressor, or an insulator, located within the deletion, and (2) 16 sequence variants identified in the Bbs7 promoter region (36.613.319 – 36.614.267, Ensembl release 102) in the BFMI mice compared to the B6N DNA sequence which can be responsible for differences in Bbs7 gene regulation. This study aimed to answer two main questions: (1) Does the deleted intron 8 region of Bbs7 which includes a CTCF-element have any partial or complete impact on the development of juvenile obesity in BFMI mice and what is the explanation for that? (2) What is the causal variant in the promoter region of the Bbs7 gene that leads to expression differences of the Bbs7 gene between BFMI and B6N? Both, the single 5’ UTR-SNP and the deletion in intron 8 of Bbs7 can contribute to the observed phenotype in the BFMI mice most likely by reducing Bbs7 expression and increasing fat accumulation

A 5′ UTR Mutation Contributes to Down-Regulation of Bbs7 in the Berlin Fat Mouse

The Bardet–Biedl Syndrome 7 (Bbs7) gene was identified as the most likely candidate gene causing juvenile obesity in the Berlin Fat Mouse Inbred (BFMI) line. Bbs7 expression is significantly lower in the brain, adipose tissue, and liver of BFMI mice compared to lean C57BL/6NCrl (B6N) mice. A DNA sequence comparison between BFMI and B6N revealed 16 sequence variants in the Bbs7 promoter region. Here, we tested if these mutations contribute to the observed differential expression of Bbs7. In a cell-based dual-luciferase assay, we compared the effects of the BFMI and the B6N haplotypes of different regions of the Bbs7 promotor on the reporter gene expression. A single-nucleotide polymorphism (SNP) was identified causing a significant reduction in the reporter gene expression. This SNP (rs29947545) is located in the 5′ UTR of Bbs7 at Chr3:36.613.350. The SNP is not unique to BFMI mice but also occurs in several other mouse strains, where the BFMI allele is not associated with lower Bbs7 transcript amounts. Thus, we suggest a compensatory mutation in the other mouse strains that keeps Bbs7 expression at the normal level. This compensatory mechanism is missing in BFMI mice and the cell lines tested

A deletion containing a CTCF-element in intron 8 of the Bbs7 gene is partially responsible for juvenile obesity in the Berlin Fat Mouse

The Berlin Fat Mouse Inbred (BFMI) line is a model for juvenile obesity. Previous studies on crosses between BFMI and C57Bl/6N (B6N) have identified a recessive defect causing juvenile obesity on chromosome 3 (jObes1). Bbs7 was identified as the most likely candidate gene for the observed effect. Comparative sequence analysis showed a 1578 bp deletion in intron 8 of Bbs7 in BFMI mice. A CTCF-element is located inside this deletion. To investigate the functional effect of this deletion, it was introduced into B6N mice using CRISPR/Cas9. Two mice containing the target deletion were obtained (B6N Bbs7emI8∆1 and Bbs7emI8∆2) and were subsequently mated to BFMI and B6N to generate two families suitable for complementation. Inherited alleles were determined and body composition was measured by quantitative magnetic resonance. Evidence for a partial complementation (13.1–15.1%) of the jObes1 allele by the CRISPR/Cas9 modified B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was found. Mice carrying the complementation alleles had a 23–27% higher fat-to-lean ratio compared to animals which have a B6N allele (P(Bbs7emI8∆1) = 4.25 × 10–7; P(Bbs7emI8∆2) = 3.17 × 10–5). Consistent with previous findings, the recessive effect of the BFMI allele was also seen for the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles. However, the effect size of the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was smaller than the BFMI allele, and thus showed only a partial complementation. Findings suggest additional variants near Bbs7 in addition to or interacting with the deletion in intron 8

A 5′ UTR Mutation Contributes to Down-Regulation of Bbs7 in the Berlin Fat Mouse

The Bardet–Biedl Syndrome 7 (Bbs7) gene was identified as the most likely candidate gene causing juvenile obesity in the Berlin Fat Mouse Inbred (BFMI) line. Bbs7 expression is significantly lower in the brain, adipose tissue, and liver of BFMI mice compared to lean C57BL/6NCrl (B6N) mice. A DNA sequence comparison between BFMI and B6N revealed 16 sequence variants in the Bbs7 promoter region. Here, we tested if these mutations contribute to the observed differential expression of Bbs7. In a cell-based dual-luciferase assay, we compared the effects of the BFMI and the B6N haplotypes of different regions of the Bbs7 promotor on the reporter gene expression. A single-nucleotide polymorphism (SNP) was identified causing a significant reduction in the reporter gene expression. This SNP (rs29947545) is located in the 5′ UTR of Bbs7 at Chr3:36.613.350. The SNP is not unique to BFMI mice but also occurs in several other mouse strains, where the BFMI allele is not associated with lower Bbs7 transcript amounts. Thus, we suggest a compensatory mutation in the other mouse strains that keeps Bbs7 expression at the normal level. This compensatory mechanism is missing in BFMI mice and the cell lines tested.Peer Reviewe