Effects of post-weaning diet on metabolic parameters and DNA methylation status of the cryptic promoter in the Avy allele of viable yellow mice  Denise A. Warzak, Sarah A. Johnson, Mark R. Ellersieck, R. Michael Roberts, Xiang Zhang, Shuk-Mei Ho, Cheryl S. Rosenfeld  Journal of Nutritional Biochemistry  Volume 26, Issue 6, Pages 667-674 (June 2015) DOI: 10.1016/j.jnutbio.2015.01.003 Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 1 Photographic comparison of a Y4-Y5 yellow coat color mice before (A) and after (B) being placed on the NIHMe diet for 29 weeks. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 2 Average body weights for juvenile, early adult and adult yellow and brown coat color Avy/a males and females on the different post-weaning diets. (A) Male weights; (B) female weights. Data are reported as the average±S.E.M. Two comparisons are represented in the graph: (1) comparing different coat color animals fed the same diet and (2) comparing same coat color animals within diets. Superscripted letters, such as a,b, demarcate differences between diets (AIN, NIH or NIHMe) for animals of the same coat color groups (yellow or brown) (P<.05); asterisks (*) represent differences between Avy/a mice possessing different coat colors (yellow or brown) but within the same diet groups (AIN, NIH or NIHMe) (P<.05). Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 3 Average serum glucose concentrations for juvenile, early adult and adult yellow and brown coat color Avy/a males and females on the different post-weaning diets. (A) Males; (B) females. Data are reported as the average±S.E.M. Two comparisons are represented in the graph: (1) comparing different coat color animals fed the same diet and (2) comparing same coat color animals within diets. Superscripted letters, such as a,b, demarcate differences between diets (AIN, NIH or NIHMe) for animals of the same coat color groups (yellow or brown) (P<.05); asterisks (*) represent differences between Avy/a mice possessing different coat colors (yellow or brown) but within the same diet groups (AIN, NIH or NIHMe) (P<.05). Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 4 DNA methylation analysis within the IAP region Avy cryptic promoter of brown and yellow coat color male and female (n=6 for each diet and coat color) on the different post-weaning diets. (A) Percent CpG methylation for the nine CpG sites; (B) mean total CpG methylation for the IAP site in the Avy locus. *P=.01, **P=.003 and ***P<.0001. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 5 Type II regression analysis for brown coat color Avy/a mice on the AIN diet comparing Avy CpG methylation versus body weight. Asterisks (*) represent individual animals. R2=0.9421, R=−0.97 and P=.0013. There are six data points to reflect the three males and three females whose DNA methylation status was analyzed. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S1. Diagram of the viral intracisternal A particle (IAP) in the Avy gene. The IAP is embedded in pseudoexon 1a, and it contains a cryptic promoter. When this site is hypomethylated, Avy is constitutively and ectopically expressed, leading to a yellow coat color accompanied by adult-onset obesity and diabetes. However, methylation of this IAP silences the cryptic promoter site and transcription then resumes solely from the normal exon 2 start site, which is confined to the mid-phase of the hair cycle such that the animals possess a pseudoagouti (brown) coat color. Nine CpG islands, which are demarcated in boldface and underlined and located within the IAP between the cryptic Avy promoter and IAP promoter and downstream 3′ genomic sequences, were screened. IAP DNA sequence is represented in normal font, while sequence from the unmodified, adjacent part of the A gene is italicized. The boldfaced text in the A sequence represents the location of the reverse primer used to PCR amplify the Avy IAP region. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S2. Weekly to every other week body weight for brown and yellow coat color Avy/a males on the AIN, NIH and NIHMe diets. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S3. Weekly to every other week body weight for brown and yellow coat color Avy/a females on the AIN, NIH and NIHMe diets. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S4. Final adult body weight for males (A) and females (B). The final body weight was assessed when the animals were 32 weeks of age. Data are reported as the average±S.E.M. For males, asterisk (*) represents significant differences (P<.05). For females, *P<.01 and **P<.0001. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S5. Correlation between average CpG methylation in the IAP site in the Avy gene and body weight. The only significant correlation was the greater degree of DNA methylation at this IAP site in AIN brown animals inversely correlated with body weight gain. In other words, the more demethylated this site became in this group of animals, the more likely they were to gain weight. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S6. Type II regression analysis for brown coat color Avy/a mice on the NIH diet comparing Avy CpG methylation versus body weight. Asterisks (*) represent individual animals. R2=0.1656, R=−0.41 and P=.4232. There are six data points to reflect the three males and three females whose DNA methylation status was analyzed. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. S7. Type II regression analysis for brown coat color Avy/a mice on the NIHMe diet comparing Avy CpG methylation versus body weight. Asterisks (*) represent individual animals. R2=0.0016, R=−0.04 and P=.9405. There are six data points to reflect the three males and three females whose DNA methylation status was analyzed. Journal of Nutritional Biochemistry 2015 26, 667-674DOI: (10.1016/j.jnutbio.2015.01.003) Copyright © 2015 Elsevier Inc. Terms and Conditions