Volume 64, Issue 5, Pages 982-992 (December 2016) Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues  Kimberly A. Krautkramer, Julia H. Kreznar, Kymberleigh A. Romano, Eugenio I. Vivas, Gregory A. Barrett-Wilt, Mary E. Rabaglia, Mark P. Keller, Alan D. Attie, Federico E. Rey, John M. Denu  Molecular Cell  Volume 64, Issue 5, Pages 982-992 (December 2016) DOI: 10.1016/j.molcel.2016.10.025 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 982-992DOI: (10.1016/j.molcel.2016.10.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Gut Microbiota Affect Host Tissue Epigenetic States (A) Experimental design: (1) tissues harvested from germ-free (GF), conventionally raised (ConvR), and conventionalized (ConvD) mice. (2 and 3) Histones extracted, chemically derivatized, and trypsinized. (4 and 5) Histone peptides injected onto mass spectrometer and data acquired on >50 unique histone PTM states. (B) Relative abundance of histone PTMs on H3, H3.3, and H4. Values are reported as a fold change relative to GF controls (log2). Mean percentage of peptide family total across all samples (rightmost column). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4 mice per condition. Molecular Cell 2016 64, 982-992DOI: (10.1016/j.molcel.2016.10.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Gut Microbiota-Mediated Epigenetic Changes Are Sensitive to Diet (A) Experimental design: (1) GF and ConvR mice on either chow or a HF/HS (“Western”) diet. (2–5) Tissues were prepared and analyzed as described in Figure 1. (B) Weights of GF and ConvR mice fed a HF/HS diet at the time of sacrifice. (C and D) Hepatic total cholesterol (C) and hepatic triglycerides (D) in GF, ConvD, and ConvR mice on chow and HF/HS diets. (E) SCFA measurement in cecal contents from GF, ConvR, and ConvD animals on chow and HF/HS diet. ∗p < 0.05, ∗∗p < 0.01; error bars represent SD; n = 4 mice per condition. (F–H) H4 (K5, K8, K12, and K16) acetylation in colonized liver (F), adipose tissue (G), and proximal colon (H) relative to GF controls (fold change, log2). (I and J) H3 K18 and K23 methylation and acetylation in colonized liver (I) and proximal colon (J) relative to GF controls (fold change, log2). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; error bars represent SEM; n = 4 mice per condition. Molecular Cell 2016 64, 982-992DOI: (10.1016/j.molcel.2016.10.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Hepatic Genes Are Co-regulated in Colonized Mice as a Function of Diet or Colonization Status (A) K-means clustering of differentially expressed hepatic genes (left, fold change versus GF, log2) and KEGG pathway enrichment terms (right). (B–D) Interaction network for cluster 2 (B), cluster 4 (C), and cluster 6 (D). Only genes with at least one reported interaction are graphed. Edges indicate interaction. Node size indicates relative expression in HF/HS-fed mouse livers. Node color indicates relative expression in chow-fed mouse livers. Molecular Cell 2016 64, 982-992DOI: (10.1016/j.molcel.2016.10.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 SCFA-Supplementation Mimics Colonization-Induced Epigenetic Programming (A) Experimental design: (1) GF mice were supplemented with SCFAs (GF + SCFA) or colonized (ConvD), and tissues were harvested. (2–5) Histone extracts were prepared as described in Figure 1. (B) Hierarchical clustering of histone PTMs in ConvD and GF + SCFA mouse tissues (fold change versus GF, log2). (C and D) Pearson’s correlation of ConvD and GF + SCFA mouse tissue histone PTM states in liver (C) and proximal colon (D). (E) K-means clustering of differentially expressed hepatic genes in ConvD and GF + SCFA mice. FDR cut-off for differential expression = 0.05; n = 3 mice per condition. (F) GO term enrichment in clusters b and c. (G) Overlap of differentially expressed genes between ConvD and GF + SCFA mice. Molecular Cell 2016 64, 982-992DOI: (10.1016/j.molcel.2016.10.025) Copyright © 2016 Elsevier Inc. Terms and Conditions