Volume 140, Issue 2, Pages 572-582.e2 (February 2011) Variants in RET Associated With Hirschsprung's Disease Affect Binding of Transcription Factors and Gene Expression  Yunia Sribudiani, Marco Metzger, Jan Osinga, Amanda Rey, Alan J. Burns, Nikhil Thapar, Robert M.W. Hofstra  Gastroenterology  Volume 140, Issue 2, Pages 572-582.e2 (February 2011) DOI: 10.1053/j.gastro.2010.10.044 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 An overview of the reported HSCR-associated SNPs. Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Schematic overview of the RET gene and the constructs used in this study. (A) SNP rs2435357 (Enh1:C>T) and SNP rs2506004 (Enh2:C>A) are both located in a conserved region of intron 1, called MCS+9.7, separated by 217 base pairs. (B) RET and SV40 promoter constructs coupled to luciferase and enhancer sequences used in this study. Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 (A) Under SV40 promoter regulation, both Enh1-T and Enh2-A separately or in combination decreased luciferase expression approximately 2-fold. (B) Under RET promoter regulation, only when both disease-associated SNPs were present could they decrease luciferase expression. (C) Constructs with the RET promoter coupled to the enhancer sequence containing the combination of a non–disease-associated variant and its disease-associated counterpart (Enh1-T/Enh2-C and Enh1-C/Enh2-A) showed that both SNPs could significantly decrease luciferase expression. The bars indicate the mean ± SEM of the fold changes of luciferase expression for each construct. P < .05 was considered significant. Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 A and B show EMSA using labeled oligonucleotides containing Enh2-C and Enh2-A with NPE of Neuro2A cells transfected with NXF/ARNT2, NXF, or ARNT2 constructs; an extra band (white arrow) appeared when oligonucleotides containing Enh2-C allele were used in combination with NPE containing both NXF and ARNT2. In a competition assay (Cp-Sp) in lane 5, the extra band as seen in lane 1 disappeared, while excess of unrelated, unlabeled oligonucleotide (Cp-NS) could not compete with the specific labeled oligonucleotide (lane 6). (C and D) EMSA-supershift (SS) assays using Enh2-C and Enh2-A labeled oligonucleotide with total cell lysates of the SK-N-SH cell line and anti-NXF antibody showed that the highest band was shifted upwards and the second lower band became weaker (was blocked) (lane 4). Based on C, the highest band (white arrow), which is shifted by the NXF antibody, is the NXF/ARNT2 complex, and the second band (black arrow), for which the binding is blocked in lane 4, most likely is NXF. (E and F) EMSA using labeled oligonucleotides containing Enh2-C and Enh2-A with NPE of Neuro2A transfected with SIM2/ARNT2, SIM2, or ARNT2 construct. SIM2 has the same binding affinity to the enhancer sequences containing Enh2-C or Enh2-A (lanes 1 and 3); the specificity of this binding is confirmed in competition specific and nonspecific assays (lanes 5 and 6). Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 (A) Using reverse-transcription PCR, it is shown that RET, NXF, SIM2, and ARNT2 are expressed in NCSCs isolated from mouse embryonic gut: E11.5, 12.5, 13.5, 14.5, and 15.5. (B) Progenitor status of RET-positive cells in NLB cultures by coimmunostaining with the neural crest marker p75: (i) DAPI staining, (ii) RET expression, (iii) p75 expression, and (iv) coexpression of RET and p75. (C) SIM2 coexpression with RET in NLB cultures: (i) DAPI staining, (ii) RET expression, (iii) SIM2 expression, and (iv) coexpression of RET and SIM2. (D) ARNT2 coexpression with RET in NLB cultures: (i) DAPI staining, (ii) RET expression, (iii) ARNT2 expression, and (iv) coexpression of RET and ARNT2. Scale bars = 100 μm in B (iv) and 50 μm in C (iv) and D (iv). Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 (A) The effect of NXF/ARNT2 on luciferase expression of constructs containing the RET promoter or the RET promoter and enhancer containing Enh2-C or Enh2-A. (B) The effect of SIM2/ARNT2 on luciferase expression of constructs containing the RET promoter or the RET promoter and enhancer containing Enh2-C and Enh2-A variants. (C and D) A possible (not significant) dosage effect of NXF/ARNT2 and SIM2/ARNT2 on luciferase expression driven by the RET promoter and enhancer regulation. Three dosages of NXF/ARNT2 and SIM2/ARNT2 constructs were used. The bars indicate the mean ± SEM of fold changes of luciferase expression for each construct after cotransfection of NXF/ARNT2 or SIM2/ARNT2. In C and D, the bars indicate the mean ± SEM of normalized luciferase expression for each construct after cotransfection with 3 different dosages of NXF/ARNT or SIM2/ARNT2. P < .05 was considered significant. Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 (A) Western blotting of total cell lysate of Neuro2A cells transfected with 3 different dosages of NXF/ARNT2 or SIM2/ARNT2, D1 (100/50 ng), D2 (500/250 ng), and D3 (1000/500 ng), using anti-RET and anti–β-actin primary antibodies. (B) Densitometric analysis of the Western blots. The highest dosage of NXF/ARNT2 enhanced RET expression 2.25-fold compared with RET expression in the mock controls. (C) Densitometric analysis of the Western blots. The highest dosage of SIM2/ARNT2 decreased RET expression 2.6-fold compared with the RET expression in the mock controls. The bars indicate the mean ± SEM of fold changes for RET endogenous expression after transfection of NXF/ARNT2 or SIM2/ARNT2. P < .05 was considered significant. Gastroenterology 2011 140, 572-582.e2DOI: (10.1053/j.gastro.2010.10.044) Copyright © 2011 AGA Institute Terms and Conditions