5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation

Santiago Uribe-Lewis,Anna Nicholson,Thomas Carroll,Suraj Menon,Adele Murrell,Piotr J Manasterski,Douglas J Winton,Simon J A Buczacki

doi:10.1038/s41598-019-57214-z

Santiago Uribe-Lewis, Anna Nicholson + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-57214-z

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Journal: Scientific Reports	Publication Date: Jan 17, 2020
Citations: 18	License type: open-access

Affiliation: University of Cambridge, University of Bath

Abstract

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Highlights

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes
Post-transcriptional events influencing the stability or activity of epigenetic factors cannot be discerned by RNA-seq, these results show that intestinal differentiation involves a complex balance in the levels of a considerable number of epigenetic factors (Supplementary Table S8)
Given the peak size and p value cut-off used to select peaks with 5hmC change (Supplementary Fig. S6) the analysis showed CpG-dinucleotide-containing motifs emerged from loci that gained 5hmC and CA repeat sequences predominant at regions with 5hmC loss

Summary

Introduction

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. In this study we have mapped gene expression and 5hmC in purified stem progenitors and differentiated epithelium of the adult mouse intestine to identify which 5hmC-marked genes play a role in intestinal differentiation. 5hmC is low in the proliferating gut crypt progenitors and increased in the differentiated villus epithelium of the mouse small intestine (SI). (d) The genomic profiles of 5hmC and how they may correlate with gene activity upon gut differentiation were assessed by profiling expresssion (RNA-seq) and 5hmC (hmeDIP-seq) of the purified crypt stem progenitors (Cd24a_Mid) and differentiated villi (Cd24a_Neg) Ulex-lectin was used to deplete differentiated Paneth and Goblet cells and Cd45 to remove hematopoietic cells. (d) The genomic profiles of 5hmC and how they may correlate with gene activity upon gut differentiation were assessed by profiling expresssion (RNA-seq) and 5hmC (hmeDIP-seq) of the purified crypt stem progenitors (Cd24a_Mid) and differentiated villi (Cd24a_Neg)

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