Epigenetics and Obesity

1. Epigenetics and Obesity A potential role for the imprinted gene MEST in diet-induced obesity in mice Robert A. Koza Department of Molecular Genetics Pennington Biomedical Research Center

2. epigenetics Factors contributing to the current obesity epidemic genetics environment

3. Body weights measured weekly 3 wks 8 wks 12 wks weaned; chow diet high fat diet, NMR NMR, tissue collection Experimental design used for feeding study with 112 male C57BL/6J mice

4. Bodyweight variation primarily due to increased adiposity

5. Strong association of MEST with adiposity, but not lean mass (112 mice HEF 23) Microarray Analysis High versus low weight gaining mice; HFD for 2, 4 and 12 weeks Identified a gene called mesoderm specific transcript(MEST/Peg1) to be more highly expressed in adipose tissue of high weight gaining mice. Microarray data was validated by qRT-PCR

6. Adipose tissue MEST is induced in some, but not all mice by dietary fat in as little as 2 days Epididymal Inguinal

7. Mesoderm Specific Transcript (MEST) AKA Peg1 (paternally expressed gene 1); mouse Chr 6 (7.5 cM); human 7q32; function unknown, putative soluble epoxide (a/b) hydrolase (lipid transport and metabolism?). Protein of 335 amino acids (~37 kDa) Targeted mutation leads to abnormal maternal behavior (impaired placentophagia) and growth retardation (L. Lefebvre et al, 1998). Overexpression in mouse adipose tissue and 3T3-L1 adipocytes leads to increased adipocyte size and and induced expression of adipogenic transcription factors (Takahashi et al, 2004) Importance implicated in mammalian metanephrenic development (Kanwar et al. 2002) and oncofetal angiogenesis (Mayer et al. 2000). Loss of imprinting (LOI) implicated in etiology of lung adenocarcinoma, colon cancer and metastatic breast cancer (Kohda et al. 2001; Nishihara et al. 2000; Pederson et al. 1999, 2002). Several reports of alternative splicing, transcript variants and possibly an antisense transcript.

8. Bisulfite Sequencing Is variability in adipose tissue MEST expression due to differences in methylation of the MEST gene?

9. Animals selected for CpG methylation analysis of inguinal fat DNA Mouse# MEST (AU/ng RNA) 12 wk BWT FM/LM (12 wk) 496 0.45 29.1 0.19 501 1.15 32.1 0.25 530 1.20 32.9 0.25 549 0.55 28.8 0.18 552 1.62 30.6 0.19 587 0.68 30.80.20 Mean= 0.94 ± 0.19 30.72 ± 0.66 0.21 ± 0.013 498 38.31 31.1 0.38 524 30.84 34.0 0.39 537 30.75 32.0 0.41 560 42.76 34.6 0.38 562 42.83 34.4 0.38 573 32.3333.0 0.36 Mean= 36.30 ± 2.34 33.18 ± 0.57 0.38 ± 0.006 p<10-7 p=0.02 p<10-5

10. DNA imbedded in agarose beads m -TTTGGCGAATTT- -CTCGGCGAACCC- Methodology: DNA isolated from mouse inguinal fat Denaturation with NAOH Bisulfite conversion of non-methylated cytosine to cytosine sulfonate Hydrolytic deamination converts cytosine sulfonate to uracil sulphonate Alkali desulphonation to convert uracil sulphonate to uracil PCR with GSPs/TOPO-TA cloning/BDT sequencing

11. …showed no differences in methylation of MEST gene in inguinal fat of high vs low MEST expressing mice Methylation Analysis -analysis of 22 CpGs within a 318 bp DMR region 5’ from open reading frame

12. 498 524 537 560 562 573 496 530 501 549 552 587 HIGH MEST EXPRESSION_Region B analyses LOW MEST EXPRESSION_Region B analyses

13. Summary ->98% of cytosines not found in CpGs were effectively converted to uracils -clear patterns of methylation were observed in maternal vs paternal alleles -no significant differences in adipose tissue MEST promoter (CpG island) DNA methylation were apparent between low and high MEST expressing mice

14. MEST is induced by dietary fat in adipocytes but not stromal-vascular fraction of fat depot

15. TSS TSS ORF ORF A B C 22 22 CpGs CpGs 31 31 CpGs CpGs 15 15 CpGs CpGs 0 0 +334 +334 +92 +92 - - 175 175 - - 1100 1100 +116 +116 - - 818 818 (N=38) (N=38) AD AD AD AD (N=37) (N=37) 80 80 SV SV 80 80 SV SV (N=46) (N=46) (N=28) (N=28) 70 70 70 70 (N=62) AD Region C Region A Region B Region C SV (N=65) 60 60 60 60 80 Region B 50 50 50 50 70 40 40 40 40 60 % methylation 30 30 30 30 50 20 20 20 20 40 10 10 10 10 30 0 0 0 0 20 15 15 1 1 31 31 1 1 10 CpG Islands CpG Islands CpG Islands CpG Islands 0 1 22 CpG Islands Genomic methylation patterns of MEST in mature adipocytes and corresponding stromal-vascular cells MEST Promoter

16. A B C % methylation 1 15 22 31 1 1 CpG CpG CpG Adipose tissue (ING fat) from low vs high MEST expressing mice MEST Promoter

17. Region C Region A % methylation % methylation 1 31 15 1 CpG methylation in adipocytes isolated from epididymal fat of mice with high or low MEST expression after 7 days on HFD MEST Promoter

18. MEST vs Sfrp5 MEST vs Nkd1 Sfrp5 vs Nkd1 Summary Loss of imprinting is observed in genomic regions of adipocyte DNA that are ~1 kb upstream from, and immediately distal to the characterized imprinted genomic region of the MEST promoter. No differences in methylation patterns were observed in total inguinal fat DNA, or epididymal fat adipocyte DNA from mice with high or low MEST mRNA expression. These data suggest that the MEST promoter in adipocytes is ‘poised’ for activation by an undefined mechanism. Future studies will examine genes that are highly associated with MEST expression in adipose tissue. These will include several genes involved in Wnt signaling.

19. Acknowledgements PBRC Les Kozak Larissa Nikonova Jessica Hogan Jong-Seop Rim Tamra Mendoza Chris Faulk Ken Eilertsen Jihad Skaf Applied Biosystems HEF- L. Kozak, D. York CNRU- E. Ravussin