Short-chain fatty acids propionate and butyrate control growth and differentiation linked to cellular metabolism [CUT&Tag]

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. Multiomic profiling (ChIP/ATAC/RNA-seq and CUT&Tag) was performed on short-chain fatty acids propionate and butyrate in colorectal cancer (CRC) and normal cell lines (SW480, CCD841, CT26) and on dietary fiber-supplemented (5% arabinoxylan) mouse intestines to determine their regulatory function. Cells were treated with 0 - 10 mM sodium propionate or sodium butyrate overnight and next-generation sequencing libraries were prepared according to individual sequencing type protocols. N = 2 experimental replicates were prepared per condition/IP for each histone mark and the corresponding control in ChIP-seq and CUT&Tag, plus n = 2 input for ChIP-seq. Tissues from two male CETP-ApoB-100 transgenic mice procured from Taconic Biosciences (New York, USA) were used per condition. N = 3 experimental replicates were prepared for ATAC/RNA-seq in SW480 cells. N = 4 experimental replicates were prepared for ATAC-seq in CT26 cells. For ChIP-seq and CUT&Tag genome-wide profiling of propionyl and butyryal histone marks in cells and in vivo, corresponding acetyl marks were used as controls for differential binding analysis. For ATAC/RNA-seq on treated cells (1 or 10 mM for propionate and butyrate, respectively) untreated (0 mM) conditions were used as controls. Next-generation sequencing was performed on Illumina HiSeq 4000 and NovaSeq 6000 SP platforms using paired-end, single index sequencing. Raw data was then analyzed for differential binding and accessibility, pathway and motif enrichment and differential expression to gain insight into the regulatory role of SCFA in the gut epithelium in the context of CRC and normal cells, as well as in relation to dietary fiber supplementation in vivo.