Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts

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1 Cell Stem Cell Supplemental Information Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts Jurian Schuijers, Jan Philipp Junker, Michal Mokry, Pantelis Hatzis, Bon-Kyoung Koo, Valentina Sasselli, Laurens G. van der Flier, Edwin Cuppen, Alexander van Oudenaarden, and Hans Clevers

2 A B figure S1 Density β catenin ASCL2 TCF4 Paneth Cells 16% 14% 12% 1% 8% 6% 4% p=8* distance from closest TSS (kb) 2% % VillinCreERT2 Ascl2 -/- C Relative expression level Rnf43 Lgr5 Cbx6 Aqp1 Stem cell Other [IWP-2] µm Figure S1. Ascl2 and β-catenin/tcf4 co-occupy DNA elements in mouse intestinal crypts and drive a stem cell program. Related to figures 1 and 3. A) Ascl2, β-catenin and Tcf4 peak distribution relative to transcription start sites in mouse intestinal crypts. Peaks from ChIP-seq data were accumulated around all transcription start sites from the RefSeq database. Related to figure 1. B) Percentage of Paneth cells per micrograph. VillinCreert2 and Ascl2 loxp/loxp organoids were harvested 3 days after exposure to 4OHT, sectioned and immunohistochemistry for Lysozyme was performed in combination with H&E staining. Nuclei were used to quantify the total amount of cells per micrograph, Lysozyme positive Paneth cells were scored and the % of Paneth cells per micrograph was determined. More than 1 micrographs were used per cell type. The average % of Paneth cells per micrograph is plotted. Error bars = standard deviation. Related to figure 3. C) qpcr analysis of IWP-2 treated organoids. Wild type organoids were exposed to different concentrations of IWP-2 porcupine inhibitor for 24 hours and mrna was quantified for a panel of Wnt-target genes that are considered stem cell markers, Lgr5 and Rnf43, as well as Wnt target genes that are not in the stem cell signature: Aqp1 and Cbx6. Error bars = standard deviation. Related to figure 3.

3 figure S2 A.4.3 Tcf4 KO Ascl2 Rescue.2.1 NES=1.77 P>.1 Enriched in Lgr5 HI cells Enriched in Lgr5 LO cells Figure S2 Genes rescued by Ascl2 over-expression correlate with stem cell genes. Related to figure 4. GSEA probing for enrichment of a Ascl2-rescued gene set in Lgr5 HI stem cells versus Lgr5 LO cell expression analysis. Gene set exist of genes that are up-regulated >1.5 fold when Ascl2 is over-expressed in a Tcf4 -/- background. The used gene list was obtained from a comparison of the gene expression from sorted Lgr5 HI versus Lgr5 LO daughter-cells. Red = genes enriched in Lgr5 HI stem cells. Blue = genes enriched in Lgr5 LO daughter cells.

4 A T T A 38 1 kb Ascl2 figure S3 36 β-catenin 154 TCF4 B RNF43 ASCL2 TCF4 C ASCL2 motif C CCT C G CC G T G C G G T G 359 β-catenin D Control β-catenin ASCL2 β-cat & ASCL2 E 8 Top1-luciferase reporter activity RLU 2 RLU Figure S3. Chip-seq shows DNA co-occupation by ASCL2, β-catenin and TCF4 in Ls174t colon cancer cells. Related to figure 4. A) Example of the ChIP-Seq patterns of ASCL2, β-catenin and TCF4, RNF43 locus displayed. Only the signal around called peaks is shown. B) Venn-diagram showing the overlap of ASCL2, β-catenin and TCF4 binding sites. C) Sequence logo depicting the E-box DNA binding motif de novo identified in ASCL2 peaks. D) Luciferase reporter assay testing for synergetic activation of TCF4-ASCL2 cooccupied elements in 293T cells. Reporters were co-transfected with ASCL2, S33-βcatenin or both. Luciferase activity was normalized with the value obtained from the pgl4.1 backbone without enhancer. Triplicates of a representative experiment are shown. E) Luciferase reporter assay using the TOP1 control reporter for Wnt activity. TOP1 consists of 1 consensus TCF motifs in front of a TATA box driving the luciferase gene. This reporter was co-transfected with S33-β-catenin, ASCL2 or both. Luciferase activity was normalized with the value obtained from the pgl4.1 backbone without enhancer. Triplicates of a representative experiment are shown.

5 A figure S4 RLU MYB enhancer Control β-catenin Ascl2 β-catenin & Ascl2 1 5 con mt 1 mt 2 mt bp Ascl B Transcript density (*1-3 transcripts per μm 2 ) Tcf4 1 2 Ascl2 mrna abundance Time (hours) Figure S4. Wnt signaling and Ascl2 form a bi-stable switch for stemness. Related to figure 5 and 6. A) Luciferase reporter assay in HEK293T cells showing loss of transcriptional activation of the MYB enhancer upon mutagenesis of ASCL2 or TCF4 motifs. The location of the mutated motifs and the ChIP-seq pattern over the cloned enhancers are shown in the bottom panel (numbers). CON = the unaltered MYB enhancer. 1 = mutated Ascl2 motif #1. Black bar indicates the cloned region. RLU = relative luciferase units. Luciferase activity was normalized to the value obtained from the pgl4.1 backbone without enhancer. Triplicates of a representative experiment are shown. Related to figure 5. B) Single molecule FISH quantification of Ascl2 signal after withdrawal of R-spondin CM. Images were taken and cropped to contain only cells. These images were then rasterized in pseudo cells of 25 µm 2 each and dots per pseudo cell were counted. The mean transcript density (dots per µm 3 ) was plotted. Error bars = SEM. Related to figure 6.

6 figure S5 A Phalloidin-GFP DAPI % R-spondin 4% R-spondin Cbx6-Cy5 B 16x1-3 Transcript density (molecule per µm3) Addition Withdrawal 12x1-3 8x1-3 4x Figure S5. The Wnt responsive gene Cbx6 does not show hysteresis. Related to figure 6. A) Fluorescence microscopy showing single molecule fluorescence in situ hybridization for Cbx6 (Cy5) mrna in organoid cultures. Individual dots represent single mrna molecules. Cell membranes have been stained by GFP-coupled phalloidin and nuclei by DAPI. Scale bars 12.5µm. White dashed lines indicate a single cell. B) Single molecule FISH quantification of mrna molecules per cubic micrometer. Organoid cultures were cultured in the presence or absence of 5% R-spondin CM for 16 hours before the medium was changed to contain different amounts of R-spondin CM. After 16 hours, organoids were collected and processed to cryo-sections before being submitted to single molecule FISH. Images were taken and cropped to contain only cells. These images were then rasterized in pseudo cells with an xy area of 25 µm2 each and dots per pseudo cell were counted. The mean transcript density (dots per µm3) was plotted. Solid lines show a sliding window average, taking the average of three adjacent data points centered on the middle of the three. Error bars = SEM.

7 A 13 figure S6 Fold enrichment over control region wt α-ascl2 sirna 1 A B C D E F A B C D E A B C D E F A B C D 1st quartile 2nd quartile 3rd quartile 4th quartile B Relative Luciferase units control Ascl2 over-expression 2 1 LGR5 enhancer I LGR5 enhancer II MYB enhancer KLHDC4 enhancer SMOC2 enhancer SOX9 enhancer I SOX9 enhancer II SLC7A5 enhancer OSBPL5 enhancer EPHB3 enhancer KIAA 1199 enhancer PTPRO TSS BMP7 enhancer PCK1 TSS NR2E3 TSS Figure S6. ASCL2 binds to DNA and ASCL2 bound elements activate target genes. Related to Experimental Procedures. A) ChIP-PCR analysis of 21 control elements for Ascl2 binding in the presence and absence of sirna targeting Ascl2. Control elements were selected from the four quartiles of Ascl2 peaks based on peak intensity. Values depicted are means of three qpcr reactions from the same ChIP experiment. Enrichment was normalized using a control region in the β-globin locus. B) Luciferase reporter assay of Asscl2 bound elements in 293T cells in the presence of absence of a co-transfected Ascl2 expression vector. Luciferase activity was normalized with the value obtained from the pgl4.1 backbone without enhancer. Triplicates of a representative experiment are shown. Error bars = standard deviation.

8 Table S1. Peaks called for ChIP-seq of Human ASCL2, Mouse Ascl2 and Mouse β-catenin. Related to figure 1. The ChiP library was sequenced on SOLiD/AB sequencer to produce 5- bp long reads. Sequencing reads were mapped against the reference genome. Cisgenome was used for peak-calling with.1 FDR.

9 Table S2. C(t) values of different TCF/LEF family members. Related to figure 4. Sample Lef1 Tcf1 Tcf3 Tcf4 Organoid N/A Thymus mes Table S3. Regulatory region motif analysis of the Ascl2 locus in mouse and human. Related to figure 6. Human Promoter Mouse Promoter Rank Motif Score Motif Score 1 StuAp PHD h GATA Blimp SOK Xbp TBS PHD Blimp SOK isx GATA USF MEF-2C 1.4 STAT FACB.984 StuAp STF1.777 MEF-2C TGA1b.731 TGA1b ECM23.74 HSF GATA SFP TBS1.662 HBP-1a HBP-1a.659 FACB UF1H3BET.641 UF1H3BET HSF2.628 TGA1b TGA1b.619 CPRF Adf NeuroD CPRF CPRF Human 5'UTR Mouse 5' UTR Rank Motif Score Motif Score 1 RXRGdime Blimp IXR GATA hkb MEF-2C ATF PHD StuAp SOK Su(H) CEP Pit HSF Su(H) STB SMAD Xbp AZF MIZF Six ARGRI SREBP ARG BarbieBox MEIS Zta p Blimp EHF AZF Tcfcp2l SREBP Spz btd RGM TBP R.42 2 Pax TBS1.414

10 Supplemental Experimental Procedures primer sequences for enhancer cloning of Luciferase reporter plasmids Enhancer Cloning primer 5' Chromosome location Length LGR5 intronic enhancer 5' AACTATCTTAAAGTCCAGATCCC chr12:7,135,123-7,135, LGR5 intronic enhancer II 5' TTAGATAAGTCCACATAAAGACGGG chr12:7,136,2-7,137,1 868 PCK1 promoter 5' GCATATAATCTAGCAAAACTGCC chr2:55,568,568-55,569, NR2E3 promoter 5' GCCTGTAATCCCACCTGATTGGG chr15:69,889,55-69,89, KLHDC4 intronic enhancer 5' CTGCATGTCACGGGACAATGCTCG chr16:87,766,298-87,767,7 773 MYB enhancer 5' CAGAGGATGCTGTATAGTAAGCG chr6:135,629, ,63, KIAA1199 intronic enhancer 5' TCTCTGTCACAGCTCCCTGGCCC chr15:78,936,48-78,936, SMOC2 intronic enhancer 5' CTAGATATGCTAAAATCAGGGTGG chr6:168,739, ,74, EPHB3 enhancer 5' CCTCAGCCTGGAAACAGGAAGGG chr3:185,759,65-185,76, PTPRO promoter 5' GAGAAAGGAAGACAGGTTGTTGGC chr12:15,59,3-15,591,1 76 BMP7 intronic enhancer 5' GTCAGGTCTTGCTGAGCGCTGGG chr2:55,22,842-55,221, SOX9 enhancer 5' GTGTGCATCTGTGTTGAGGTGGC chr17:67,65,77-67,651, LGR5 intronic enhancer 3' TTTATGAAGTTAGCACTACTGGG chr12:7,135,123-7,135, LGR5 intronic enhancer II 3' GCCTCCTTTATTCCCCTACCATAGGC chr12:7,136,2-7,137,1 868 PCK1 promoter 3' CATTCGACGGCCTTTTGCCAGGGC chr2:55,568,568-55,569, NR2E3 promoter 3' ACCGTGTTTCTAGGATTCTGGG chr15:69,889,55-69,89, KLHDC4 intronic enhancer 3' CTGTTCAGCCTGATGTTGCAGCCC chr16:87,766,298-87,767,7 773 MYB enhancer 3' CAGGTAGCTTAAGACTTAACACCC chr6:135,629, ,63, KIAA1199 intronic enhancer 3' CTGCTCTAAGACACCTTCTGCCC chr15:78,936,48-78,936, SMOC2 intronic enhancer 3' CTAGGACCTCATATTGCCATGTGC chr6:168,739, ,74, EPHB3 enhancer 3' GGTCAGAGACAGAGGCATGTGTGGG chr3:185,759,65-185,76, PTPRO promoter 3' CAACTTTTGGGCTTGGTCTAAGCC chr12:15,59,3-15,591,1 76 BMP7 intronic enhancer 3' AGCTTTAATTTTAGCCTTCCTGG chr2:55,22,842-55,221, SOX9 enhancer 3' GACACTCATAGAAGGAAGAGACCC chr17:67,65,77-67,651,55 799

11 qpcr primers used for ASCL2 ChIP-seq validation Group Primerpair Forward Chromosome location 1 Random_peak1A 5' GGCAGGTGCAAAGCAAATCCTATC chr11:3,12,5-3,121,2 Random_peak1B 5' AGGTGTTAAGGAGAACACAGGTGC chr5:11,2-11,8 Random_peak1C 5' TATAGCACGGCCTCGTTCAACAGT chr8:143,421,8-143,422,3 Random_peak1D 5' ACAGATGTGTGTGCAGGTGCATGA chr21:45,678,2-45,678,6 Random_peak1E 5' CTGTTCTTTGGGACTTGTCCAGCA chr17:71,417,234-71,417,661 Random_peak1F 5' TGCCTTCTCAAAGTGCTGGGATCA chr2:233,87, ,871,342 2 Random_peak2A 5' GTTTGCACAGCTGCTCTGGATTGT chr21:42,68,528-42,68,792 Random_peak2B 5' AAATGAGTCAGCTAGGCAGAGGCT chr1:199,419,1-199,419,317 Random_peak2C 5' CTCCATCTGACAGGAAAGGGAACA chr1:6,465,7-6,466, Random_peak2E 5' TCCAGCCAATGACAGCCTATGGAA chr8:14,76,536-14,76,878 Random_peak2F 5' AGAAACCTGGAGCTCACCACTCTT chr19:52,843,428-52,843,677 3 Random_peak3A 5' ACTGGCAAACCTATAGCGAGGCAA chr14:27,269,277-27,269,55 Random_peak3B 5' TGACCGGTAAGACTGGTTCCTGGT chr22:3,69,4-3,69,6 Random_peak3C 5' ACACAGGTGTAACCTGACAAAGCC chr14:75,12,729-75,13,129 Random_peak3D 5' TCCTGACAAAGGAAACTGCCTCCA chr11:93,912,288-93,912,527 Random_peak3E 5' AGGCTTCAGGTGACTGCAGAAGAT chr17:43,889,55-43,889,854 Random_peak3F 5' CATTCATGATCTCTCCACAGGCTC chr7:64,314,22-64,314,454 4 Random_peak4A 5' AGGTGCAGCTCACGTGATGTAAGT chr2:19,58,762-19,59,332 Random_peak4C 5' ACAGGTGGGCCTGCTTGAAAGATA chr6:169,942,7-169,943,46 Random_peak4D 5' TCCCGACGTCACATGATTCTCCAA chr17:39,777,773-39,778,6 Random_peak4E 5' ACTGGTCTGGTTCTTGAAGGTGGA chr1:14,31,58-14,31,939 Random_peak4F 5' TGTCTGTGGCAAGTCAGTGAACCT chr5:141,911,45-141,911,31 1 Random_peak1A 3' ACCACCCATGTAGAAGCTGGGTTA chr11:3,12,5-3,121,2 Random_peak1B 3' ACCTGTTCTAGTTCCCGCCTTCAT chr5:11,2-11,8 Random_peak1C 3' TAGGCATGGCGTCTGAGCTCTT chr8:143,421,8-143,422,3 Random_peak1D 3' TTGCACAGGAAGTGTCTGCTCA chr21:45,678,2-45,678,6 Random_peak1E 3' ACAGAGGTTTCTCTGATCCTCCTC chr17:71,417,234-71,417,661 Random_peak1F 3' TCCTGGTGCTGGAAACAGGTATGA chr2:233,87, ,871,342 2 Random_peak2A 3' AAAGGCATGCAGGAGAGAACAGGA chr21:42,68,528-42,68,792 Random_peak2B 3' GCACTTGGCACTGTGCAATTCTCT chr1:199,419,1-199,419,317 Random_peak2C 3' CTTCAGGAATCTGGGTCATCCTGT chr1:6,465,7-6,466, Random_peak2E 3' TTTCACCTGCCATTCCGCATCCT chr8:14,76,536-14,76,878 Random_peak2F 3' AGTCCCTCTCCTGCTTGCTTTACT chr19:52,843,428-52,843,677 3 Random_peak3A 3' TTACTGTTGGCATGCCTGAAACCC chr14:27,269,277-27,269,55 Random_peak3B 3' CCTGGATTGTAGGACCTTGTGTGT chr22:3,69,4-3,69,6 Random_peak3C 3' TTTCCATATGGGCAAGTGCCAAGG chr14:75,12,729-75,13,129 Random_peak3D 3' AAGGCAGTTGAGTCCCTTCCTACA chr11:93,912,288-93,912,527 Random_peak3E 3' CGCTTCGAGAAAGCGGAAAGAAGT chr17:43,889,55-43,889,854 Random_peak3F 3' ACAAGTCCAGACACACCTGGAGAA chr7:64,314,22-64,314,454 4 Random_peak4A 3' AATTCAACTCGGCGAGAAGGCAGA chr2:19,58,762-19,59,332 Random_peak4C 3' ACACACAAGGCAGAGAGAGAGAGA chr6:169,942,7-169,943,46 Random_peak4D 3' AAATCACTCTCCTCCCTGCTTCCT chr17:39,777,773-39,778,6 Random_peak4E 3' AAGCAAACCACAGAGCCTTAGGTG chr1:14,31,58-14,31,939 Random_peak4F 3' TGGACTGGGCTTTGGTACTGTGAT chr5:141,911,45-141,911,31

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