Gut Microbiota Affect Host Tissue Epigenetic States

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McCoy K.D.

Macpherson A.J. Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota. Krautkramer et al., 2015 Krautkramer K.A.

Reiter L.

Denu J.M.

Dowell J.A. Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry. Figure 1 Gut Microbiota Affect Host Tissue Epigenetic States Show full caption (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 (log 2 ). 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. To investigate whether gut microbes and their metabolites affect host chromatin states, we examined histone PTM states as a function of colonization. We focused our analysis on proximal colon, liver, and white adipose tissue (WAT). The experimental workflow is described in Figure 1 A. Briefly, mice were either maintained germ-free (GF) throughout the experiment, allowed to acquire a microbiota from birth to adulthood (conventionally raised, ConvR), or colonized with a complete (uncultured) microbiota (conventionalized, ConvD) harvested from ConvR donors. Use of a ConvD mouse model allows for the determination of whether the phenotype observed in ConvR animals is transferrable via the gut microbiota alone. Additionally, since ConvR animals experience different environmental exposure early in life and have developmental differences () that may exhibit phenotypic differences versus their GF controls, the use of ConvD mice for relatively short time periods allows for dissection of effects more directly related to differences in microbial metabolism. Histones were extracted from tissues and prepared for mass spectrometry analysis using an in-house workflow ().

We surveyed 55 unique and combinatorial acetylated and methylated histone PTM states in proximal colon, liver, and WAT ( Table S1 ). Colonization induced robust increases in H4 acetylation in all three tissues ( Figure 1 B). This peptide includes the first 4 lysines (K5, K8, K12, and K16) on the H4 N-terminal tail. Thus, H4: 0ac indicates peptides where no lysine residues are acetylated, whereas H4: 1ac–4ac indicates peptides where any 1–4 of the 4 lysines are acetylated. In ConvR animals, there was a significant 2.1-fold increase in both triply and quadruply acetylated H4 (H4: 3ac and H4: 4ac, respectively). Similarly, ConvR animals showed a 3-fold and 1.30-fold increase in the highly acetylated H4: 4ac peptide in proximal colon and adipose tissue, respectively, relative to GF mice ( Figure 1 B). The effects of colonization on H4 acetylation were even more robust in ConvD mice, with a 4.5-, 6.0-, and 12.0-fold increase in H4: 4ac of proximal colon, adipose, and liver, respectively ( Figure 1 B). Triply acetylated H4 peptides also increased 2.1- to 4.2-fold in proximal colon, adipose, and liver ( Figure 1 B). It is noteworthy that these two H4 states collectively account for just over 1% of the total H4 states, suggesting that this open chromatin state is confined to very specific loci along the genome. Consistent with the conversion of unacetylated states to higher acetylation, the completely unmodified form decreased significantly (H4: 0ac, 1.33- to 3.3-fold across tissues surveyed) in colonized animals.

Microbes similarly induced acetylation of canonical H3 and the variant H3.3. The doubly acetylated canonical H3 K9ac + K14ac and H3 K18ac + K23ac peptides increased significantly in ConvD mouse livers and trended toward a similar magnitude increase in proximal colon of ConvR and ConvD mice ( Figure 1 B). These two doubly acetylated canonical H3 peptides account for roughly 2% or less of total histone PTM states observed in each peptide family, again supporting a more loci-specific role for these modified nucleosomes along the genome ( Figure 1 B). Interestingly, the singly acetylated peptides K9ac + K14un and K9un + K14ac decrease concomitantly with an increase in the doubly acetylated K9ac + K14ac peptide, and a similar pattern occurs on the singly acetylated and coeluting K18ac/K23ac peptides ( Figure 1 B). These results are consistent with a shift away from a singly acetylated state toward a maximally acetylated state.

H3 methylation patterns are also altered as a function of gut colonization status. There is a modest, yet statistically significant increase in H3 K27me3 + K36un in proximal colon, liver, and adipose tissues from ConvD mice versus their GF controls (1.4- to 1.5-fold increase; Figure 1 B), accounting for ∼12% of total PTM states and suggesting more broad regulatory effects in comparison to highly acetylated states of H3 and H4. There were increases in peptides containing highly methylated forms of K27 and K36 (i.e., me2 and me3) on both the canonical H3 and the variant H3.3; however, these effects were not present consistently across all three tissues, suggesting some tissue specificity in the response to colonization ( Figure 1 B). Notably, H3 K18me1 decreased across all three ConvD tissues (2.1- to 8.3-fold across liver, proximal colon, and adipose tissue; Figure 1 B). A similar pattern was present for the combinatorial K27me2 + K36me1 peptide on H3 and variant H3.3 ( Figure 1 B). Together, these results demonstrate that gut microbiota affect host tissue acetylated and methylated chromatin states in a site-specific and combinatorial fashion, strongly supporting a role for the gut microbiota as a driver of host tissue chromatin regulation. While some histone PTM states appear to be similarly regulated across all tissues surveyed, other changes are unique within a tissue.