ChIP-Seq-Based Approach in Mouse Enteric Precursor Cells Reveals New Potential Genes with a Role in Enteric Nervous System Development and Hirschsprung Disease

Abstract

Hirschsprung disease (HSCR) is a neurocristopathy characterized by intestinal aganglionosis which is attributed to a failure in neural crest cell (NCC) development during the embryonic stage. The colonization of the intestine by NCCs is a process finely controlled by a wide and complex gene regulatory system. Several genes have been associated with HSCR, but many aspects still remain poorly understood. The present study is focused on deciphering the PAX6 interaction network during enteric nervous system (ENS) formation. A combined experimental and computational approach was performed to identify PAX6 direct targets, as well as gene networks shared among such targets as potential susceptibility factors for HSCR. As a result, genes related to PAX6 either directly (RABGGTB and BRD3) or indirectly (TGFB1, HRAS, and GRB2) were identified as putative genes associated with HSCR. Interestingly, GRB2 is involved in the RET/GDNF/GFRA1 signaling pathway, one of the main pathways implicated in the disease. Our findings represent a new contribution to advance in the knowledge of the genetic basis of HSCR. The investigation of the role of these genes could help to elucidate their implication in HSCR onset.

Keywords: ChIP-seq; Hirschsprung disease; PAX6; gene expression profiling; sequence analysis.

Figure 1

Genes with different expression levels in Hirschsprung disease (HSCR)-NLBs. (A) Genes that showed different expression levels between HSCR-NLBs and control NLBs. Analysis of differential gene expression of potential susceptibility genes for HSCR. (B) Analysis of differential gene expression relative to a fold change. (C) Heat map representative of the results. The heat map was generated using DataAssist v3.0 software (Life Technologies) and it represents the messenger RNA expression levels of such genes expressed in colon tissue from HSCR patients and controls. Genes were hierarchically clustered by Pearson correlation coefficient using average linkage. The color scale, representing ΔCt, is shown on the right side. Green indicates genes with relatively decreased expression in HSCR, whereas red indicates genes with relatively increased expression in HSCR compared with the controls. * p value < 0.05, ** p value < 0.01 and *** p value < 0.001.

Figure 2

Functional role of Grb2 in NLB cultures. (A) Grb2 expression on mouse enteric precursor cells’ (EPCs) Grb2 downregulation. Uninfected group (control), small hairpin RNA (shRNA) non-target control group (LV-off-target), and infected group (LV-Grb2). p-value control/LV-Grb2 = 0.0017; LV-off-target/LV-Grb2 = 0.02. (B) Effect of LV-Grb2 on the size and number of NLB cultures. (C) Effects of LV-Grb2 on the cell phenotypes derived from NLBs (EPCs that express Nestin, Beta-tubulin III, or p75 markers). For Nestin, p-value control/LV-Grb2 = 0.002; LV-off-target/LV-Grb2 = 0.003. A representative image of the decrease of Nestin-expressing cells (PE-CF594-A+) in LV-Grb2 condition detected by flow cytometry is shown on the right side. Fluorochrome APC-A corresponds to Beta-tubulin III marker. * p value < 0.05, ** p value < 0.01 and *** p value < 0.001.

Figure 3

Diagram that shows the workflow with the selection, prioritization, and further validation of the candidate genes.

Figure 4

Diagram that shows the methodological approach used for the selection of PAX6 target genes.