Dnmt3b knock-down in enteric precursors reveals a possible mechanism by which this de novo methyltransferase is involved in the enteric nervous system development and the onset of Hirschsprung disease.

Ana Torroglosa,Salud Borrego,Guillermo Antiñolo,Raquel María Fernández,María José Moya-Jiménez,Leticia Villalba-Benito

doi:10.18632/oncotarget.22473

Ana Torroglosa, Salud Borrego + Show 4 more

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https://doi.org/10.18632/oncotarget.22473

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Abstract

Hirschsprung disease (HSCR, OMIM 142623) is a pathology that shows a lack of enteric ganglia along of the distal gastrointestinal tract. This aganglionosis is attributed to an abnormal proliferation, migration, differentiation and/or survival of enteric precursor cells (EPCs) derived from neural crest cells (NCCs) during the enteric nervous system (ENS) embryogenesis. DNMT3b de novo methyltransferase is associated with NCCs development and has been shown to be implicated in ENS formation as well as in HSCR. In this study we have aimed to elucidate the specific mechanism underlying the DNMT3b role in such processes. We have performed the knockdown of Dnmt3b expression (Dnmt3b-KD) in enteric precursor cells (EPCs) to clarify its role on these cells in vitro. Moreover, we have analyzed several signaling pathways to determine the mechanisms responsible for the effect caused by Dnmt3b-KD in EPCs. Our results seem to support that Dnmt3b-KD promotes an increase EPCs proliferation that may be mediated by P53 and P21 activity, since both proteins were observed to be down-regulated in our Dnmt3b-KD cultures. Moreover, we observed a down-regulation of P53 and P21 in HSCR patients. These results lead us to propose that DNMT3b could be involved in HSCR through P53 and P21 activity.

Highlights

Hirschsprung disease (HSCR, OMIM 142623), the most common neurocristopathy in humans (1:5000 newborns), is characterized by the absence of enteric ganglia along variable lengths of the distal gastrointestinal tract, resulting in severe intestinal dysfunction [1]
First we evaluated the expression of Dnmt3b in the Neurosphere like-bodies (NLBs) cultures by SYBR green real-time RT-PCR technique
We analyzed the possible effect of Dnmt3b-KD in the NLBs cultures and observed a significant increase of NLBs number in KD compared with the control (C) and small hairpin RNA (shRNA) Non-Target Control (Non-Target) conditions (Figure 1A)

Summary

Introduction

Hirschsprung disease (HSCR, OMIM 142623), the most common neurocristopathy in humans (1:5000 newborns), is characterized by the absence of enteric ganglia along variable lengths of the distal gastrointestinal tract, resulting in severe intestinal dysfunction [1]. Based on the length of the aganglionic region, HSCR phenotypes are classified as: short-segment forms (S-HSCR) which include patients with aganglionosis as far as the splenic flexure, long-segment forms (L-HSCR) in which aganglionosis extends beyond the splenic flexure and total colonic aganglionosis forms (TCA) [2]. Such aganglionosis is attributed to a failure of the proliferation, migration, differentiation and/or survival of the enteric precursors cells (EPCs) derived from NCCs during embryonic development of Enteric Nervous System (ENS). One well characterized epigenetic mechanism regulating gene expression is DNA methylation, which in mammalian is mediated by three DNA methyltransferases: DNMT1, DNMT3a and DNMT3b [5]. DNMT3a and DNMT3b function as de novo methyltransferases and together are responsible for methylation pattern acquisition during gametogenesis, embryogenesis, and somatic tissue development as well as for maintaining the silence of transposable elements and enhancing the stability of genome. [6, 7]

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