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1 Supplementary Materials for Disruption of histone methylation in developing sperm impairs offspring health transgenerationally Keith Siklenka, Serap Erkek, Maren Godmann, Romain Lambrot, Serge McGraw, Christine Lafleur, Tamara Cohen, Jianguo Xia, Matthew Suderman, Michael Hallett, Jacquetta Trasler, Antoine H. F. M. Peters,* Sarah Kimmins* *Corresponding author. (S.K.); (A.H.F.M.P.) This PDF file includes: Figs. S1 to S9 Table S1 Legends for Tables S2 to S7 Published 8 October 21 on Science Express DOI: /science.aab26 Other Supplementary Material for this manuscript includes the following: (available at Tables S2 to S7

2 Supplementary Figure 1. Transgenic copy number was determined by Southern blot and expression in embryonic tissues was not detected. (a) Transgenic construct and southern-blot probe location (b) Copy number was assessed by Southern blot followed by densitometry using spiked samples for transgenic (TG) lines 1 and 2. (c) Localization of the transgene marker GFP in a cryostat cross-section from adult testis. (d-e) Total RNA from transgenic embryos at E18. was assessed for transgene expression from testis, liver, kidney, gut, lungs, heart, and brain. PCR amplification of cdna with primers against hskdm1 did not show transgene expression in these tissues. Positive controls include cdna from adult transgenic testis and purified plasmid. Gel image was acquired using Qiagen s Qiaxcel Advanced. Supplementary Figure 2. (a) Sperm counts for line1 TG F2 (TG 2 ; n=1), nontg (nontg 2 ; n=), and for line2 TG F 2 (TG 2 ; n=4), nontg (nontg 2 ; n=3), and control (n=7). (b) Body weight (BW), (c) Testes weight to body weight (TW:BW), (d) Epididymal weight to body weight ratio (EW:BW), for TG and nontg animals of Line1 (F 1 = TG 1 n=8; F 2 = TG 2 n=26, nontg 2 n=9; F 3 = TG 3 n=11, nontg 3 n=9), and Line2 (F 1 = TG 1 n=3, nontg 1 n=3; F 2 = TG 2 n=6, nontg 2 n=6; F 3 = TG 3 n=3; F 4 = nontg 4 n=4). (e) Fetus bodyweight and (h) placenta weight at embryonic day 18. (E18.) for embryos sired by control C7BL/6 (n=1), TG (n=12) and nontg (n=11) fathers. Differences between TG and nontg groups were compared to control with student s T-test corrected for multiple comparisons by Bonferroni corrections. Values depicted by mean ± standard deviation. Supplementary Figure 3: (a) Panels i, ii: Histopathological analysis of spermatogenesis was performed on testis cross-sections from generations TG 2-3 and nontg 2-3 in comparison to controls. Panels iii, iv: DNA damage did not differ in TG and nontg germ cells as assessed by normal immunohistochemical staining for phosphorylated gamma H2AX on testis cross-sections from TG 2-3 (n=4) versus C7BL/6 (n=3). (b) RNA from whole testis of C7BL/6 (n=2), TG (n=3) and nontg (n=3) were quantified via qpcr to measure the dct between Line1 UTR (forward: -GGCGAAAGGCAAACGTAAGA-3 ; reverse: - GGAGTGCTGCGTTCTGATGA-3 )(6) and Gapdh gene (forward: -ACTTTGGCATTGAAGGGCTG-3 ; reverse: TGGAAGAGTGGGAGTTGCTGTTG-3 ) (Students t-test, p. =.2.) (c) Number of tunnel positive tubules and (d) cells per 1 tubules in testes from adult C7BL/6 (n=4) and TG 1 males (n=4). 1

3 Supplementary Figure 4. Variable offspring outcome in litters sired by individual males and analyzed between PND -21. Genotypes and generations are: a) C7BL/6 b) Line 1 TG 2 c) Line 1 TG 3 d) Line 1 nontg 3 e) Line 2 TG 2, f) Line 2 TG 3, and g) Line 2 TG 4 Supplementary Figure : Wild-type C7BL/6 skeletal anatomy and normal staining patterns at embryonic day 18.. (a-d) Alcian blue stains cartilage (blue), and alizarin red stains ossified bone (red). Supplementary Figure 6. (a) Pair-wise scatter plots show the correlation of total library size normalized read counts (log2) at the region ± 2 bp surrounding TSS for H3K4me2 in sperm of C7BL/6 males (two replicates), KDM1a TG 3 and nontg 3 littermates. Genes which have more than or equal to reads (log2 scale) in the regions analyzed in both replicates of H3K4me2 BL6 are shown in red. (b) Density plot showing the ratio of H3K4me2 in sperm of KDM1A TG 3 males over C7BL/6 males in regions ± 2 bp surrounding transcriptional start sites (TSS). The plot area is divided into 3 zones by green lines. The zone on the left specifies the genes with reduced H3K4me2, the middle zone specifies the genes with unchanged H3K4me2, and the right zone shows the genes gaining H3K4me2. For the specification of the green lines, please see materials and methods. Supplementary Figure 7. Heatmaps showing nucleosome occupancy around TSS (±3kb) in sperm of C7BL/6 WT, KDM1A TG 3 and nontg 3 littermates. Genes were classified in three groups according to H3K4me3 and H3K27me3 states in WT sperm(17). H3K4me2 occupancies around TSS of corresponding chromatin samples are shown in Figure b and c. Mono-nucleosomes isolated from MNase digested sperm chromatin (input). Supplementary Figure 8. DNA methylation analysis by quantitative Sequenom MassARRAY, Mean ± SEM of methylation (%) at individual CpGs for regions of high H3.3, CpG density and major depletion of H3K4me2 (ratio of TG vs WT < -3). No differences were detected between methylation levels in transgenic (TG 3 ), non-transgenic (nontg 3 ) and controls sperm. 2

4 Supplementary Figure 9. Analysis of RRBS in sperm of TG 3, nontg 3 and control animals. (a) Heatmap of Pearson s correlation coefficients between all pairs of samples, calculated using percent methylation of CpGs with a minimal coverage of 1 reads (n =.64 million). Similar samples were grouped by hierarchical clustering as shown by dendrogram. (b) To identify genotype associated methylation, the methylation status of each individual CpG was summarized in a contingency table with the rows corresponding to methylated and unmethylated reads, and columns to three genotypes. A P-value was calculated for each CpG using Fischer s exact test. The figure shows the number of P-values below a given cut-off (p ) for the observed data (black dots) and expected by chance (red line). At p = 1/ (number of CpG) we find 629 significant CpGs. (c) Heatmap of percent methylation for 629 CpGs with significant genotype associations. CpGs with similar methylation levels across multiple samples were grouped by hierarchical clustering as shown by dendrogram. The average difference of methylation between groups is typically small (< 2%). Furthermore, there is no overrepresentation of methylation patterns that correlate with transgenerational phenotypes. 3

5 A) Probe for Southern Ef1α K Flag hskdm1 cdna Intron IRES EGFP BGH 26 bp B) EcoRI EcoRI TG 1 TG 2 WT 1X 1X X 1X C) Merge TG 1x 1x C7BL/6 1x 1x D) hskdm1a TG E18. TG Adult Testis Liver Kidney Gut Lungs Heart Brain Testis Plasmid Water Size (bp) Testis Liver Kidney Gut Lungs Heart Brain Testis Plasmid Water E) Gapdh TG E18. TG Adult Size (bp) Supplemental Figure 1

6 A) C7BL/6 18 TG 16 nontg 14 C) Sperm count (cells x1 / ml) TW:BW (ra9o) F2 F2 Line 1 Line 2 * * F1 F2 F3 F1 F2 F3 F4 E) E18. body weight (g) Line 1 Line EW:BW (ra;o) B) D) Body weight (g) F) Placenta weight (g) F1 F2 F3 F1 F2 F3 F4 Line 1 Line 2 F1 F2 F3 F1 F2 F3 F4 Line 1 Line Supplemental Figure 2

7 A) C7BL/6 TG B) i iii SC SC ii iv SC SC ddct C7BL/6 TG nontg Genotype C) # Tunnel positive tubules /1 tubules adult_alpha Adult L1 C7BL/6 TG 1 D) 14 # Tunnel positive cells/1 tubules adult splitted: Adult ctr_alpha_beta L1 Supplemental Figure 3

8 A) MG-344 B) Sire ID C) Sire ID MG-286 MG-279 MG-277 MG-274 MG-273 MG-271 MG-27 MG-268 MG-192 MG-181 MG-133 MG-132 MG-131 MG-13 MG-129 MG TG 739TG 633TG 63TG 69TG 18TG 17TG 16TG 1TG 49TG 494TG 49TG 48TG 47TG 2 4 #Pups #Pups (3) D) E) F) G) () 1 2 Sire ID Sire ID 268TG 184TG 183TG 177TG 174TG 162TG 19TG 1TG 14TG 98TG 768wt 62wt 636wt 63wt 626wt 624wt 623wt 617wt 68wt 493wt 492wt 481wt (3) 2 4 #Pups #Pups (4) (4) (4) (4) () (3) () Abnormal Dead Survived 92TG 422TG (4) 6TG Sire ID 91TG 79TG (3) (3) Sire ID 411TG 38TG 326TG 32TG (4) Sire ID 44TG 473TG 471TG 49TG 324TG 47TG #Pups 2 4 #Pups 1 #Pups Supplemental Figure 4

9 A) B) Interparietal Parietal Supraoccipital Frontal Exoccipital Basioccipital Arcus post atlantis (C1) Cervical vertebrae (C1-C7) Clavicula Premaxilla Scapula Arcus ant atlantis Hyoid Mandible Sternebrae (6) C) D) Temporal bones (3) Tympanicum Humerus Lumbar vertebrae (6) Femur Ilium Ischium Pubis Ulna Tibia Radius Carpals, metacarpals, phalanges Ribs (13) Fibula Thoracic vertebrae (13) Tarsals, metatarsals, phalanges Supplemental Figure

10 A) C7BL/6 H3K4me2 rep1 C7BL/6 H3K4me2 rep2 KDM1A TG 3 H3K4me2 nontg 3 H3K4me2 B) Density KDM1A TG 3 / C7BL6 (log2) Supplemental Figure 6

11 H3K4me2 down C6BL/6 TG3 H3K4me2 unchanged nontg3 C7BL/6 TG3 nontg3 H3K4me3 high H3K4me3 intermediate H3K27me3 high Supplemental Figure 7

12 A) Hnrpdl C7BL/6 TG 3 nontg 3 (chr: ) B) Grwd (chr7: ) C) Slc2a (chr1: Supplemental Figure 8

13 D) Irs2 C7BL/6 TG 3 nontg chr8: E) Klf CpG Location chr: F) Fbxl chr11: Supplemental Figure 8

14 G) Enoph1 C7BL/6 TG 3 nontg chr: to H) Aebp CpG Location chr6: I) Znrf chr8: Supplemental Figure 8

15 J) Sox11 (1 of 2) C7BL/6 TG 3 nontg 3 chr12: K) Sox11 (2 of 2) chr12: L) Arid1b chr17: Supplemental Figure 8

16 M) Zbtb2 C7BL/6 TG 3 nontg chr12: N) Hnrnpk chr13: O) Cdh chr18: Supplemental Figure 8

17 P) Zswim6 C7BL/6 TG 3 nontg chr13: Q) Fam117b chr1: R) AI chr4: Supplemental Figure 8

18 S) Pard C7BL/6 TG 3 nontg 3 chr8: T) Pde8a chr7: U) Nudt chr17: Supplemental Figure 8

19 V) Atp2b C7BL/6 TG 3 nontg 3 (chr1: Supplemental Figure 8

20 A) B) C) Supplemental Figure 9

21 Supplementary Table 1 External observations on pups based on visible appearance up to post natal day 21. Sire Line 1 Line 1 Line 1 Line 1 Line 1 TG 2 TG 3 nontg 3 nontg 4 nontg #Sires #Litters #Pups Litter size ± stdev 7.±1.8*** 7.6±2.73* 8.78± ± 2.9* 8.7±3.3 Total Abnormal Pups 71*** 21*** (dead and alive) Live Abnormal pups % 28.*** 17.4*** Dead w/ & w/o ext. malf 34.9** 62.*** 26.6* 11.4 % Sire Line 2 Line 2 Line 2 TG 2 TG 3 TG 4 #Sires 4 6 #Litters 1 14 #Pups Litter size ± stdev 7.6± ± ±1.79 Total Abnormal Pups 17*** 2*** 7*** (dead and alive) Live Abnormal pups % 18.4*** 16.3*** 17.9** Dead w/ & w/o ext. malf *** 76.9*** % Sire Control C6BL/6 #Sires 18 #Litters 26 Pups 238 Litter size ± stdev 9.1±1.71 Total Abnormal Pups 9 (dead and alive) Live Abnormal pups % 3.78 Dead w/ & w/o ext. malf 8.82 % *p<.;**p<.1;***p<.1 (statistical significance in comparison to control; Student s T-test) TG = transgenic where superscript denotes generation from founder. nontg 3 =has a transgenic father, nontg 4 =TG grandfather nontg =TG great-grandfather. * abnormal=pup showed an external abnormality. Abnormalities included growth retardation (<7% of average body weight of the litter), limb defect (for example extra or missing digits), craniofacial deformity, failure to thrive, skin abnormalities.

22 Legends for Supplementary Tables S2-S7 (Excel files) Table S2: Summary of skeletal abnormalities observed in a subset of samples from transgenic and non-transgenic E18. fetuses. (Abnormality: ; decrease in ossification/underdeveloped: O; extra point(s) of ossification: + O; missing point(s) of ossification: -O; absent structure: A.) Table S3: H3K4me2 depleted genes enriched in high (cluster 1), or intermediate (cluster 2) levels of H3K4me3, or H3K27me3 (cluster 3) in TG 3 sperm Table S4: GO Terms of H3K4m2 depleted or unchanged regions enriched in high, or intermediate levels of H3K4me3, or H3K27me3 Table S: Analysis of affymetrix data showing differential RNA content in sperm from TG 3 and nontg 3 males in comparison to C7BL/6 controls Table S6: Analysis of Affymetrix array data showing differentially regulated RNA from 2-cell embryos sired by either TG 8, nontg 8, or C7BL/6 control males Table S7: Two cell embryo sample collection and array design