The microbiota programs DNA methylation to control ...10.1038... · control intestinal homeostasis...
Transcript of The microbiota programs DNA methylation to control ...10.1038... · control intestinal homeostasis...
Articleshttps://doi.org/10.1038/s41564-019-0659-3
The microbiota programs DNA methylation to control intestinal homeostasis and inflammationIhab Ansari1, Günter Raddatz2, Julian Gutekunst2, Meshi Ridnik1, Daphne Cohen1, Monther Abu-Remaileh1, Timur Tuganbaev3, Hagit Shapiro3, Eli Pikarsky4, Eran Elinav 3, Frank Lyko 2,5 and Yehudit Bergman 1,5*
1Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Jerusalem, Israel. 2Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany. 3Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel. 4The Lautenberg Center for Immunology, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Jerusalem, Israel. 5These authors jointly supervised this work: Frank Lyko, Yehudit Bergman. *e-mail: [email protected]
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NATuRE MICRoBIoLoGY | www.nature.com/naturemicrobiology
a
b
CNV
CNV/DSSGF/DSSGF
PC1: 58% Variance
PC2:
18%
Var
ianc
e
Supplementary Figure 1. Characterization of untreated and DSS-treated crypt intestinal epithelial cells (IECs).a, Representative flow cytometry gating strategy applied for sorting colonic IEC populations, using the expression of EpCAM and CD45, from germ free (GF), conventional (CNV), DSS-treated GF (GF/DSS) and DSS-treated CNV (CNV/DSS) intestinal tissues. b, Principal component analysis of crypt transcriptomes clearly separates GF (n=3), GF/DSS (n=3), CNV (n=3) and CNV/DSS (n=3) samples.
Unstained
Only CD45
Only EpCAM
CNV
Post sort
CD45 gate EpCAM gate CD45 gate EpCAM gate
CNV/DSSGF/DSS
GF GF CNV
CNV/DSSGF/DSS
CD45 & EpCAM
CD45 & EpCAM
Sorting strategy for GF and GF/DSS Sorting strategy for CNV and CNV/DSS
Supplementary Table 1. RNA-seq sample table
Sample Yield Raw reads Reads
after QC Mapped reads
CNV4 5,404,923,292 53,514,092 53,431,692 51,518,274
CNV5 5,573,506,533 55,183,233 55,098,642 53,043,083
CNV6 6,038,997,858 59,792,058 59,714,434 57,840,394
GF2 3,880,310,775 76,084,525 76,028,942 71,160,000
GF3 3,940,889,136 77,272,336 77,194,646 75,388,406
GF4 4,259,739,963 83,524,313 83,464,724 80,912,445
CNV/DSS16 6,684,363,214 66,181,814 66,102,257 63,713,510
CNV/DSS17 5,551,871,727 54,969,027 54,901,893 52,938,870
CNV/DSS18 5,604,321,229 55,488,329 55,421,402 53,227,126
GF/DSS4 3,875,679,669 75,993,719 75,926,062 74,193,414
GF/DSS5 4,079,862,963 79,997,313 79,926,121 78,103,597
GF/DSS6 4,317,708,042 84,660,942 84,597,692 82,755,402
Supplementary Table 2. Expression levels of selected genes.
CNV GF Im
mu
ne c
ell
mark
ers
Gata2 <0.1 <0.1
Meis1 <0.1 <0.1
Nfe2 0.40 0.20
Tal1 <0.1 <0.1 Mnda <0.1 0.10
Egr2 0.10 0.80
Sox5 <0.1 <0.1
Rora <0.1 <0.1
Stat4 <0.1 0.10
Lef1 0.20 0.40
IL9R 0.10 <0.1
CD3E 0.20 1.50
EM
Epcam 872.20 921.80
Cdh1 313.70 245.20
Average gene expression levels were calculated by Cuffdiff 2.0, all expression levels
represent FPKM values. Levels >1 indicate expression.
Supplementary Table 4. ChromHMM analysis.
GF 2 GF 3 CNV 4 CNV 5
E1 0.773 0.747 0.746 0.745 Heterochromatin
E2 0.238 0.234 0.224 0.221 Repressed
E3 0.061 0.062 0.058 0.057 Poised enhancers
E4 0.538 0.526 0.513 0.511 Primed enhancers
E5 0.698 0.679 0.679 0.677 Enhancer (intragenic)
E6 0.571 0.559 0.552 0.553 Active enhancers (intragenic)
E7 0.391 0.386 0.369 0.369 Primed enhancers
E8 0.071 0.072 0.065 0.065 Active enhancers
E9 0.081 0.084 0.076 0.075 Active promoter
E10 0.023 0.024 0.023 0.023 Poised promoters
E11 0.062 0.065 0.061 0.06 Active/weak promoters
E12 0.017 0.017 0.016 0.016 Canyon promoter
E13 0.322 0.319 0.302 0.3 Marginal segment
E14 0.666 0.647 0.652 0.648 Enhancer (intragenic)
E15 0.87 0.842 0.859 0.857 Transcribed gene bodies
Numbers indicate segment-specific methylation ratios.
Supplementary Table 5. Expression levels of selected genes.
CNV CNV/DSS Im
mu
ne c
ell
mark
ers
Gata2 <0.1 <0.1
Meis1 <0.1 0.10
Nfe2 0.40 0.20
Tal1 <0.1 <0.1
Mnda <0.1 <0.1
Egr2 0.10 <0.1
Sox5 <0.1 <0.1
Rora <0.1 <0.1
Stat4 <0.1 <0.1
Lef1 0.20 0.20
IL9R 0.10 <0.1
CD3E 0.20 0.30
EM
Epcam 872.20 633.96
Cdh1 313.70 174.57
Average gene expression levels were calculated by Cuffdiff 2.0, all expression levels
represent FPKM values. Levels >1 indicate expression.
Supplementary Table 6. Number of LMRs identified in CNV and CNV/DSS datasets.
All LMRs 131,133
CNV LMRs 107,976
CNV/DSS LMRs 112,236
Hyper-Methylated LMRs 20,061
Hypo-Methylated LMRs 14,585
Hyper-Methylated LMRs-Diff 17,968
Hypo-Methylated LMRs -Diff 13,800
Supplementary Table 7. ChromHMM analysis.
CNV 4 CNV 5 CNV/DSS 16 CNV/DSS 18
E1 0.746 0.745 0.695 0.7 Heterochromatin
E2 0.224 0.221 0.217 0.225 Repressed
E3 0.058 0.057 0.06 0.064 Poised enhancers
E4 0.513 0.511 0.485 0.494 Primed enhancers
E5 0.679 0.677 0.652 0.66 Enhancer (intragenic)
E6 0.552 0.553 0.528 0.535 Active enhancers (intragenic)
E7 0.369 0.369 0.347 0.354 Primed enhancers
E8 0.065 0.065 0.064 0.069 Active enhancers
E9 0.076 0.075 0.076 0.082 Active promoter
E10 0.023 0.023 0.024 0.027 Poised promoters
E11 0.061 0.06 0.058 0.062 Active/weak promoters
E12 0.016 0.016 0.015 0.017 Canyon promoter
E13 0.302 0.3 0.276 0.29 Marginal segment
E14 0.652 0.648 0.613 0.623 Enhancer (intragenic)
E15 0.859 0.857 0.835 0.841 Transcribed gene bodies
Numbers indicate segment-specific methylation ratios.
Supplementary Table 9. ATAC-seq samples and resulting peaks.
Sample Treatment Yield Raw read
pairs Coverage
Read pairs after QC
Mapped read pairs
Uniquely mapped
read pairs
Read pair duplicates
Individual peaks
ATAC1 Water 24,010,934,000 96,043,736 5.3x 60,910,590 53,696,637 46,140,602 5,201,669 214,592
ATAC2 Water 23,346,642,250 93,386,569 4.9x 53,945,854 47,234,549 40,043,381 3,970,399 224,405
ATAC4 Water 22,163,270,750 88,653,083 3.9x 41,349,154 36,018,220 29,974,211 3,205,091 292,723
ATAC5 2% DSS 24,560,173,250 98,240,693 4.1x 50,808,890 39,264,249 36,475,487 9,875,696 352,126
ATAC6 2% DSS 22,523,429,500 90,093,718 4.5x 50,681,402 43,738,695 36,327,445 5,082,880 418,483
ATAC7 2% DSS 15,576,122,000 62,304,488 3.3x 34,562,105 29,758,068 24,869,708 3,509,951 222,338
Merged overlapping ATAC peaks 391,833
Significant peaks in CNV 469
Significant peaks in CNV/DSS 21,925