Fig 1.
Exposure to a high fat diet increases Pomc methylation and reduces gene expression in the hypothalamus.
Young adult male rats were started on a high fat diet (n = 10) or standard rat chow (Control; n = 10) and remained on this diet for a total of 7 weeks. (a) Percentage change in body weight over time was greater in the high fat diet fed animals relative to controls. (b) Quantitative real-time PCR revealed that animals fed the high fat diet have reduced expression of Pomc compared to controls (n = 8–9 per group). (c) There was a significant increase in overall methylation at the promoter region of Pomc of high fat fed animals compared to controls (boxed area; n = 7–10 per group). *P < 0.05 from Control.
Fig 2.
CRISPR-dCas9-TET1 demethylation of Pomc does not prevent abnormal weight gain on a high fat diet.
(a) Schematic showing the modified CRISPR-dCas9 system where TET1 is fused to a catalytically inactive Cas9. (b) In rat B35 cell culture there was a significant increase in Pomc expression when Pomc-specific guide RNA (gRNA) was combined with the transcriptional activator dCas9-VPR (n = 3 per group). (c) The Pomc-specific gRNA developed in B and dCas9-TET1 plasmids were infused into the hypothalamus. Two weeks post infusion, animals were placed on a control or high fat diet and maintained for 7 weeks. Weight data was collected during the first 6 weeks and whole hypothalamus tissue collected at the end of the 7th week. (d-e) No significant changes were observed in body weight or percent change in body weight over time in the gRNA+dCas9-TET1 group relative to high fat fed controls. However, Pomc-gRNA+dCas9-TET1 resulted in faster weight gain relative to standard diet control diet in comparison with gRNA only high fat diet fed controls (n = 8 per group). (f) ChIP analysis revealed an increase in Cas9 enrichment at the Pomc promoter within animals infused with the dCas9-TET1 plasmid relative to similar fed control group, confirming that our dCas-TET1 complex was correctly targeted to the Pomc promoter. (g) Gene-specific (Pomc) 5-hmC methylated DNA immunoprecipitation (meDIP) analysis revealed significant increases in 5-hmC levels within the animals injected with only the gRNA and fed a high fat diet, which was lost in gRNA+dCas9-TET1 injected high fat fed animal. *P < 0.05 from Control.
Fig 3.
CRISPR-dCas9-DNMT3a methylation of Pomc does not lead to abnormal weight gain on standard or high fat diets.
(a) Schematic showing the modified CRISPR-dCas9 system where DNMT3a is fused to a catalytically inactive Cas9. (b) The gRNA developed in Fig 2 and dCas9-DNMT3a plasmids were infused into the hypothalamus. Two weeks post infusion, animals were placed on a control diet and maintained for 6 weeks. After week 6, all animals were switched to a high fat diet for the next 4.5 weeks. Body weight data were collected every 3–4 days throughout the entire diet procedure. (c-d) CRISPR-dCas9-DNMT3a targeting of Pomc did not significantly change body weight (c) or percent change in body weight (d) over the diet procedure (n = 8–12 per group).