Genome-wide analysis of DNA methylation in Hirschsprung enteric precursor cells: unraveling the epigenetic landscape of enteric nervous system development

Abstract

Background Hirschsprung disease (HSCR, OMIM 142623) is a rare congenital disorder that results from a failure to fully colonize the gut by enteric precursor cells (EPCs) derived from the neural crest. Such incomplete gut colonization is due to alterations in EPCs proliferation, survival, migration and/or differentiation during enteric nervous system (ENS) development. This complex process is regulated by a network of signaling pathways that is orchestrated by genetic and epigenetic factors, and therefore alterations at these levels can lead to the onset of neurocristopathies such as HSCR. The goal of this study is to broaden our knowledge of the role of epigenetic mechanisms in the disease context, specifically in DNA methylation. Therefore, with this aim, a Whole-Genome Bisulfite Sequencing assay has been performed using EPCs from HSCR patients and human controls. Results This is the first study to present a whole genome DNA methylation profile in HSCR and reveal a decrease of global DNA methylation in CpG context in HSCR patients compared with controls, which correlates with a greater hypomethylation of the differentially methylated regions (DMRs) identified. These results agree with the de novo Methyltransferase 3b downregulation in EPCs from HSCR patients compared to controls, and with the decrease in the global DNA methylation level previously described by our group. Through the comparative analysis of DMRs between HSCR patients and controls, a set of new genes has been identified as potential susceptibility genes for HSCR at an epigenetic level. Moreover, previous differentially methylated genes related to HSCR have been found, which validates our approach. Conclusions This study highlights the relevance of an adequate methylation pattern for a proper ENS development. This is a research area that provides a novel approach to deepen our understanding of the etiopathogenesis of HSCR. Graphic abstract

DNA methylation in necrotizing enterocolitis

“…A broader whole genome methylation analysis found that enteric precursor cells obtained from patients with HSCR exhibited an overall pattern of DNA hypomethylation compared with controls (Ref. 66). These findings are in agreement with data demonstrating reduced expression of the DNA methyltransferase DNMT3B in neural progenitor cells in the setting of HSCR (Ref.…”

The roles of non-coding RNAs in Hirschsprung's disease

“…Extensive research underscores the pivotal roles these methyltransferases assume in both intestinal and ENCCs development [ [95] , [96] , [97] ]. Villalba-Benito et al [ 98 ] observed a reduction in the overall level of DNA methylation in EPCs derived from HSCR patients. Furthermore, aberrant DNA methylation patterns were found in certain key genes associated with ENS development, such as RET, GFRA4, EDNRB, SOX10, PHOX2B, and NRG1 [ [99] , [100] , [101] , [102] ].…”

“…Furthermore, aberrant DNA methylation patterns were found in certain key genes associated with ENS development, such as RET, GFRA4, EDNRB, SOX10, PHOX2B, and NRG1 [ [99] , [100] , [101] , [102] ]. Intriguingly, Villalba-Benito et al [ 98 ] discerned that specific ncRNAs related to HSCR, such as MEG3, AFAP1-AS, IPW, NR2F1-AS1, and miR-195, exhibited aberrant DNA methylation patterns in differential methylation region (DMR) analysis. Notably, the aberrant expression of MEG3, AFAP1-AS and miR-195 has been shown to inhibit the migration and proliferation of ENCCs [ 38 , 66 , 73 ].…”

Development, Diversity, and Neurogenic Capacity of Enteric Glia

“…These small noncoding RNAs control gene expression post-transcriptionally, by base-pairing to partially complementary sequences in target messenger RNAs ( Bartel, 2004 ). Our knowledge of microRNAs and their role in ENS development is only beginning ( Kang et al, 2021 ), also mostly starting from uncovering their expression and possible roles in Hirschsprung disease ( Sergi et al, 2016 ; Villalba-Benito et al, 2021 ).…”