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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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)


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


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



E6 0.552 0.553 0.528 0.535 Active enhancers (intragenic)


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


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

Supplementary Table 10. Disease activity index (DAI) score used to evaluate the DSS-induced colitis.

Score Body weight decrease (%) Stool consistency Rectal bleeding

0 < 1 Normal Normal

1 1–5

2 5–10 Loose stools

3 10–20

4 >20 Diarrhea Gross bleeding

the microbiota programs dna methylation to control ...10.1038... · control intestinal homeostasis...

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