Volume 140, Issue 5, Pages 1547-1555.e10 (May 2011) The Nuclear Pore Complex Protein Elys Is Required for Genome Stability in Mouse Intestinal Epithelial Progenitor Cells  Nan Gao, Gangarao Davuluri, Weilong Gong, Christoph Seiler, Kristin Lorent, Emma E. Furth, Klaus H. Kaestner, Michael Pack  Gastroenterology  Volume 140, Issue 5, Pages 1547-1555.e10 (May 2011) DOI: 10.1053/j.gastro.2011.01.048 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Derivation of the conditional Elys allele. (A) Schematic of the mouse Elys locus. The Elys gene contains 36 exons. (B) Schematic diagram depicting the targeting strategy of deriving ElysloxP allele. (C) Southern blot confirms correct targeting of Elys locus in 5 independent embryonic stem cell clones (targeted allele, 9.6 kilobases; wild-type allele, 7.6 kilobases). (D) Genomic DNA polymerase chain reaction confirms genotypes of ElysloxP/loxP, ElysloxP/+, and Elys+/+ mice. The larger-molecular-weight band is amplified from the conditional allele. (E and F) Cre-mediated recombination at the Elys locus in intestinal cells was confirmed by genomic polymerase chain reaction. The primers flanking the 2 loxP sites allow amplification of a 400–base pair fragment following Cre-mediated recombination. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Intestinal progenitor cell defects in ElysloxP/loxP; Villin-Cre mice. (A) The P12 mutant mice (mut) is smaller than its wild-type littermate. Western blot analysis shows only a small amount of Elys protein (asterisk) in intestinal extracts of P7 mutants compared with wild-type (wt) littermates, whereas none is detected in the intestine of the P60 mutant. The lower band (arrowhead) is nonspecific and not consistently present. The bar graph shows the percentage of mutant and wild-type intestinal crypts with 1 or more TUNEL-positive cells (#P < .01). (B and C) TUNEL staining shows apoptotic cells (red) in mutant and wild-type intestines. White arrows point to apoptotic crypt cells in mutants. C shows a severely affected P60 crypt. TUNEL-positive cells in P60 wild-type intestine are located at the villus tip but not in crypts. (D and E) Histologic sections of P7 and P60 Elys-deficient and wild-type intestines. Arrows point to abnormal crypts in P7 mutant. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Normal NPC assembly in ElysloxP/loxP; Villin-Cre mice. (A and B) Normal perinuclear distribution of FG-Nups (green) detected by the Mab414 antibody in wild-type (wt) and mutant (mut) (A) villus epithelial cells and (B) crypt epithelial cells. Nuclei were labeled in red by propidium iodide. (C) Morphologically identical nuclear pores (white arrows) are detected in transmission electron micrographs of mutant and wild-type crypt epithelial cells. (D) Western blot shows only a very small amount of FG-Nup (95 kilodaltons) in the cytoplasmic fraction of intestinal extracts from P12 mutants. (E) Elys is coimmunoprecipitated with FG-Nups from the P12 wild-type intestine by the Mab414 antibody. Minimal levels of Elys bound to FG-Nups are detected in the mutant intestine. Levels of Nup107, an FG-Nup, serve as loading control. Input refers to intestinal extract before immunoprecipitation. (F) Western blot detects comparable levels of 3 FG-Nups recognized by Mab414 in the wild-type and mutant intestine, comparable levels of Nup107 from the intestine of P12 and P60 mutants, and reduced levels of Nup133 from the intestine of conditional P7 and P12 Elys mutant. Comparable levels of Nup133 are present in P60 wild-type and mutant intestine. H3, histone 3 loading control. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 The DNA damage response is activated in the intestine of ElysloxP/loxP; Villin-Cre mice. (A and B) Western blot detects increased levels of γH2AX and p53 proteins in whole intestinal extracts from embryonic day (E) 16.5, P7, and P60 Elys conditional mutants (mut). (C) Immunohistochemical detection of γH2AX is restricted to intestinal crypts of the P7 conditional Elys mutant but is not detected in the wild-type intestine. (D) Western blot shows normal levels of phospho-Chk1 (Ser317) and phospho-Chk2 (Ser33/35) in whole intestinal extracts of P7 and P12 conditional Elys mutants. (E and F) Western blot shows increased levels of phospho-Cdc2 (Tyr15) and phospho-ATM (Ser1981) in whole intestinal extracts of E16.5, P7, and P60 conditional Elys mutants. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Elys binds Mcm proteins in mouse intestine. (A and B) Reciprocal immunoprecipitation experiments show interaction between Mcm2 and Mcm4 and Elys in the adult mouse intestine. (A) Western blot detects Mcm2 and Mcm4 bound to Elys immunoprecipitated from intestinal nuclear extract. The red asterisk indicates band corresponding to full-length Mcm2 protein. Input refers to intestinal extract before immunoprecipitation, and -Ab refers to mock immunoprecipitation. (B) Western blot detects Elys bound to Mcm2 and Mcm4 immunoprecipitated from intestinal nuclear extract. (C and D) Levels of chromatin-bound Mcm2 and Mcm4 in P7, P15, and P60 intestinal extracts from wild-type and Elys conditional mutants. H3, Histone 3 loading control. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Deletion of Exon 3 of Elys leads to shift of open reading frame. (A) Exon 1-6 of the wild-type Elys allele. Exon 2 contains the start codon (red). 5' untranslated region is highlighted in gray. Exon 3 and its corresponding amino acid sequence are highlighted in yellow. (B) After exon 3 is excised, the recombined Elys allele contains exon 1-2 followed by exon 3-4. The predicted reading frame from this allele will result in a frame shift (8 amino acids with double-underline) after the first 40 amino acids, as well as multiple in-frame stop codons (red). Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Elys cDNA sequence derived from Cre-recombined allele in conditional Elys knockout intestines. cDNA sequence derived from recombined (floxed) and wild-type Elys alleles confirms deletion of exon 3 in the recombined (floxed) allele. The red arrow points to the transition between exons 2 and exon 4 in the recombined allele. Red asterisks denote the first inframe stop codon downstream of the exon 2 – exon 4 transition. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Growth curve of Elys conditional mutants. Data derived from individual mutant mice and individual wild-type litter mates. P6 (n=2 mutants, 2 wild type); P12 (n = 1 mutant; 8 wild types); P25 (n=1 mutant; n = 3 wild type). Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4A Small intestinal histology of Elys mutants. (A, B) Low power images of P7 wild-type (A) and mutant (B) jejunum showing altered villus and crypt morphology. (C, D) Low power images of wild-type (C) and mutant (D) ileum showing less pronounced epithelial changes. Arrows denote height of the epithelium. (E, F) Low and high power images of duodenal abnormalities in the P7 Elys mutant. Arrows point to altered crypts (E). Note Brunner glands beneath epithelium. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4B Anti-Caspase-3 positive apoptotic cells within crypts of P7 Elys mutants. Immunostains of intestinal villi and crypts from a P15 Elys mutant using anti-human/mouse activated Caspase-3 antibody (green) and anti-E-cadherin antibody (red). (A–D) Anti-activated Caspase-3 is detected in cells within crypts (arrows). (A'–D') Identical sections as A–D stained also immunostained with the anti-E-cadherin antibody to more clearly delineate epithelial morphology. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5A Apoptotic crypt cells are negative for the Paneth cell marker lysozyme. (A–D; E–H) Serial histological sections through two small intestinal crypts of a P15 Elys conditional mutant mouse. Immunostainings with anti E-Cadherin (E-Cad; green in panels A, D, E, H) and, anti-phospho-H2AX (pH2AX; red in panels B, D) and anti-Lysozyme (red in panels F, H). Dapi stained nuclei blue (panels C, D, G, H). There is no overlap between the pH2AX positive crypt cells and the Lysozyme positive crypt cells. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5B Apoptotic crypt cells are negative for the enteroendocrine cell marker Chromogranin-A. All images are immunostains of histological sections from a P15 Elys mutant. (A, C) Identical sections immunostained with either anti-Caspase-3 (green) or anti-Caspase-3 (green) and anti-E-cadherin (red) antibodies. (B, D) identical serial tissue section stained with either anti-Chromogranin-A (green) or anti-Chromogranin-A (green) and anti-E-cadherin (red) antibodies. Apoptotic cells (arrows) do not overlap with enteroendocrine cells (arrow heads). Panels A'/C' and B'/D' show similar findings. (E, G) Identical sections immunostained with either anti-γH2AX (green) alone or with Dapi (blue nuclei). (F, G). Identical serial sections immunostained with either anti-Chromogranin-A (red) alone or with Dapi (blue nuclei). Apoptotic cells (arrows) do not overlap with enteroendocrine cells (arrow heads). Panels E'/G' and F'/H' show similar findings. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 6 Elys-deficiency leads to apoptosis in embryonic and perinatal intestinal progenitors. (A–B) TUNEL staining (red) of E16.5 control and Elys-deficient intestines. (C–D) TUNEL staining (red) of P0 control and Elys-deficient intestines. Arrows in B and D point to the apoptotic cells within the intervillus epithelial pockets in mutant intestines. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 7 FG-Nup localization in Caco2 cells. (A, D) Immunostaining with the Mab414 monoclonal antibody shows that FG-Nups are localized to the nuclear envelope. (B, E) Propidium iodide staining of nuclear DNA. (C, F) Merged images of A, B and D, E respectively. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 8 Elys-deficient villus epithelial cells contain normal-appearing nuclear pores. (A–B) Transmission EM micrographs show control and mutant villus epithelial cells containing abundant nuclear pores. (C–D) High magnification images of boxed regions in A and B demonstrate that morphologically identical nuclear pores (red arrows) formed in mutant epithelial cells. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 9 Truncated protein derived from the Elys amino terminus has no effect on the intestinal epithelium of wild-type zebrafish larvae (A, B) Lateral views of live 6 day post-fertilization wild-type zebrafish larvae that had been injected with cDNA construct encoding the amino terminal amino acids encoded by the conditional allele of the mouse Elys gene, upstream of an inframe mCherry cDNA. Micro-RNA194 promoter (unpublished) drives expression in the intestinal epithelium. Elys cDNA and mCherry cDNA are separated by amino acids encoding the viral 2A peptide recognition sequence1. Post-translational processing of the fusion protein encoded by this cDNA leads to two distinct peptides; the truncated Elys and mCherry. (A) Fluorescence microscopy identified mCherry positive cells (arrows) that perforce, express the truncated Elys protein. (B) Lateral image of the identical larvae following acridine orange staining to identify apoptotic cells. Only background gallbladder fluorescence (arrow) is seen in this larvae. No apoptotic intestinal epithelial cells are detected. This larva is representative of 10 other larvae examined. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 10 γH2AX-positive cells colocalized with apoptotic crypt cells in Elys-deficient intestine. (A–D) P7 control and Elys-deficient intestines were co-stained for TUNEL (red) and γH2AX (green). (E–J) High magnification images of the boxed regions in A and B demonstrate the colocalization of TUNEL-positive and γH2AX-positive cells in mutant crypts. Nuclear DNA was labeled by DAPI in blue. Gastroenterology 2011 140, 1547-1555.e10DOI: (10.1053/j.gastro.2011.01.048) Copyright © 2011 AGA Institute Terms and Conditions