Short-chain fatty acids propionate and butyrate control growth and differentiation linked to cellular metabolism

Abstract

The short-chain fatty acids (SCFA) propionate and butyrate are produced in large amounts by microbial metabolism and have been identified as unique acyl lysine histone marks. In order to better understand the function of these modifications we used ChIP-seq to map the genome-wide location of four short-chain acyl histone marks H3K18pr/bu and H4K12pr/bu in treated and untreated colorectal cancer (CRC) and normal cells, as well as in mouse intestines in vivo. We correlate these marks with open chromatin regions along with gene expression to access the function of the target regions. Our data demonstrate that propionate and butyrate act as promoters of growth, differentiation as well as ion transport. We propose a mechanism involving direct modification of specific genomic regions, resulting in increased chromatin accessibility, and in case of butyrate, opposing effects on the proliferation of normal versus CRC cells.

Fig. 1 |

Genome-wide H3K18pr distribution. a SCFAs as regulatory elements: experimental design overview. b H3K18pr vs H3K18ac differential binding at 10 mM NaPr treatment. Sites identified as significantly differentially bound are shown in red. n = 2 experimental replicates for each mark and input. c Top ten differentially bound regions associated with H3K18pr, annotated to within 1 Kb of TSS, sorted by false-discovery rate adjusted P value (FDR < 0.05). d Top GO Biological Process, Cellular Component and Molecular Function terms of H3K18pr-associated cis-regulatory elements (5+ 1 Kb) determined by GREAT. e Differential motif analysis of H3K18pr vs H3K18ac peaks analyzed using HOMER. P values were determined by binomial test. f Normalized reads in H3K18pr vs H3K18ac-associated binding sites at 10 mM NaPr treatment. g Signal tracks for regions representing BAIAP2. Signal intensity of peaks in 95 Kb-spanning BAIAP2 region showing H3K18pr vs H3K18ac binding at 10 mM NaPr treatment with input as background.

Fig. 2 |

Genome-wide H4K12pr distribution. a H4K12pr vs H4K12ac differential binding at 10 mM NaPr treatment. Sites identified as significantly differentially bound are shown in red. n = 2 experimental replicates for each mark and input. b Top ten differentially bound regions associated with H4K12pr, annotated to within 1 Kb of TSS, sorted by false-discovery rate adjusted P value (FDR < 0.05). c Top GO Biological Process, Cellular Component and Molecular Function terms of H4K12pr-associated cis-regulatory elements (5+ 1 Kb) determined by GREAT. d Differential motif analysis of H4K12pr vs H4K12ac peaks analyzed using HOMER. P values were determined by binomial test. e Normalized reads in H4K12pr vs H4K12ac-associated binding sites at 10 mM NaPr treatment. f Distribution of H4K12pr peaks by gene type with P value measured by Chi-squared test. g Signal tracks for regions representing TTC7A. Signal intensity of peaks in 95 Kb-spanning TTC7A region showing H4K12pr vs H4K12ac binding at 10 mM NaPr treatment with input as background.

Fig. 3 |

TSS distribution profiles of H3K18ac/pr/bu and H4K12ac/pr/bu associated ChIP-seq peaks as function of read coverage. Upper panels: Aggregate read density profile plots of genomic region distributions within +/− 1 Kb of TSS as a function of log2 IP/input ratio. Lower panels: Read density heatmaps of gene distributions with maximum (z = 4) and minimum (z = - 4) values of heatmap intensities. Plots generated by deepTools.

Fig. 4 |

Propionyl and butyryl differential gene expression by RNA-seq. a Volcano plot showing gene upregulation vs downregulation in 10 mM NaPr treated vs control groups. b Hierarchical cluttering of GO ‘Biological Process’ terms of upregulated vs downregulated pathways in NaPr treated vs control groups. c Heatmaps of 50 most variable genes in NaPr treated vs control groups. d Volcano plot showing gene upregulation vs downregulation in 1 mM NaBu treated vs control groups. e Hierarchical cluttering of GO ‘Biological Process’ terms of upregulated vs downregulated pathways in NaBu treated vs control groups. f Heatmaps of 50 most variable genes in NaBu treated vs control groups. Differential expression analysis performed by DESeq2. Hierarchical clustering was performed using ShinyGO. Size of dots indicates statistically significant P values. n = 3 experimental replicates for each condition.

Fig. 5 |

Propionyl/butyryl differential RNA-seq. a Distribution of log2 CPM (counts per million) transformed expression data for all conditions. b Principal component analysis of log2 CPM transformed expression data for all conditions. c Volcano plot of propionyl vs butyryl differential expression. d Heatmaps of 50 most variable genes for all three conditions. e Hierarchical cluttering of GO ‘Biological Process’ terms of differentially expressed pathways in propionyl vs butyryl RNA-seq. Pathways are clustered together based on shared genes. Size of dots indicates statistically significant P values. Hierarchical clustering was performed using ShinyGO. n = 3 experimental replicates for each condition. Normalization of raw counts performed by ‘cpm’ analysis in edgeR. Differential expression analysis performed by DESeq2.

Fig. 6 |

Mouse butyryl ATAC-seq and Kbu CUT&Tag in mouse intestines. a Differential accessibility at 1 mM NaBu treatment. Sites identified as significantly differentially accessible are shown in red. n = 4 experimental replicates for each condition. b Top ten differentially accessible regions associated with NaBu treatment sorted by false-discovery rate adjusted P value (FDR < 0.05), and top GO ‘Biological Process’ terms associated with positive vs negative fold change. c Normalized reads in binding sites at NaBu treatment. d Correlation heatmap showing clustering of replicates from NaBu treated vs untreated group. f Top GO ‘Biological Process’ terms associated with H3K18bu in HFS + 5% arabinoxylan group. g H3K18bu and butyryl ATAC-seq annotated peak overlap. h Top GO ‘Biological Process’ terms associated with H4K12bu in HFS + 5% arabinoxylan group. i H4K12bu and butyryl ATAC-seq annotated peak overlap. Significance of overlaps determined by hypergeometric test-generated P value. j Overview of SCFAs propionate and butyrate as regulatory elements affecting histone binding, chromatin accessibility and gene expression.