A deletion in a stretch/super enhancer in the mouse 3’ region of Ank1 gene alters the expression of genes encoding proteins participating in glucose and lipid metabolism in skeletal muscle

Several Genome-wide association studies indicated an association between Single Nucleotide Polymorphisms (SNPs) in the ANK1 gene and type 2 diabetes (T2D). Interestingly, one of these SNPs is located in the second internal promoter localized in the 3’ region of the ANK1 gene. An in-depth analysis of the genomic region surrounding the second internal ANK1 promoter revealed the presence of a muscle stretch/super enhancer sequence. Stretch/super enhancers are chromatin domains characterized by a high content of gene regulatory elements capable of coordinating the transcription of different genes over large genomic distances. To evaluate whether this stretch/super enhancer participates in the regulation of the expression of genes with a potential role in T2D susceptibility, sAnk1.5 (P2) KO mice carrying a deletion around the second internal Ank1 promoter werecharacterized. These mice show increased fasting blood glucose and impaired glucose tolerance starting around 4 months of age. Transcriptomic analyses of EDL and soleus muscles revealed that pathways involved in glucose homeostasis and lipid metabolism are significantly altered in sAnk1.5 (P2) KO mice, and the alteration is muscle-type specific. The mRNA expression levels of several genes participating in glycogen, glucose, and lipid metabolism were evaluated and confirmed the transcriptomic data. To investigate the fiber-type specific alterations of KO condition, transcriptomic analysis was performed on single myofibers isolated from EDL and soleus muscles; data resulted comparable to those obtained from muscles, but a much higher number of differentially expressed genes were found, indicating that the analysis at single fiber level was much more informative, probably because of the reduction of variability present in whole tissue due to the presence of different cell types.Additionally, a fraction of muscle fibersfrom EDL of sAnk1.5 (P2) KO mice revealed a switch toward a more oxidative phenotype, indicated by the expression of slow myosin isoforms. These data represent a confirmation of the phenotype observed in sAnk1.5 (P2) KO mice from a transcriptional point of view, supporting a causative involvement of the Ank1 locus in the development of T2D. Moreover, from these preliminary data, it seems that lipid metabolism, in addition to glucose metabolism, is deeply altered, possibly indicating that skeletal muscle cells of sAnk1.5 (P2) KO mice rely more on fatty acids than glucose as an energy source.