Supplementary Figure 1 An overview of pirna biogenesis during fetal mouse reprogramming. (a) (b)
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1 Supplementary Figure 1 An overview of pirna biogenesis during fetal mouse reprogramming. (a) A schematic overview of the production and amplification of a single pirna from a transposon transcript. The transposon mrna is processed by the primary pirna biogenesis machinery to generate primary sense pirnas that have a 1U bias. Indicated in yellow is a representative pirna that will be processed from this transcript. Once bound to MILI, the pirna guides endonucleolytic cleavage of complementary transcripts between 10th and 11th nucleotide counting from a 5' end of bound pirna. The 3 cleavage product will be processed to form a secondary antisense pirna, the location of which is indicated in blue. The nascent secondary pirna is loaded into MILI or MIWI2. This licences MIWI2 to enter the nucleus, where it is proposed to tether to a nascent transposon transcript and direct de novo DNA methylation by an unknown mechanism. The binding of the secondary antisense pirna to MILI results in the pirna-guided cleavage of transposon transcript and the biogenesis of the original pirna. This MILI:MILI homotypic ping-pong cleavage cycle amplifies transposon-derived pirnas during fetal reprogramming. (b) Features of amplified pirna pairs. Due to the fact that most primary pirnas exhibit 1U bias, newly generated secondary pirnas will have a bias for A at their 10th position (10A). Complementarity between the first 10 nucleotides of amplified pirna pairs is a key signature of the ping-pong cycle.
2 Supplementary Figure 2 Loss of MILI and MIWI2 has different impact on genomic and repeat element methylation. Distribution of CpG methylation levels measured by whole-genome bisulfite sequencing of Mili -/- (dark blue) and Miwi2 -/- (light blue) spermatogonia plotted as a difference to wild-type (WT) spermatogonia (grey dotted line). Significance was tested using Wilcoxon rank sum test with Bonferroni multiple testing correction, *** indicates a p value of < Graphs show averages of values obtained from three animals. The middle line in the graphs represents median of the data points with whiskers indicating the data range.
3 Supplementary Figure 3 Transposon-related pirnas in the absence of MILI. (a) Length distribution of small RNAs in wild-type (WT) and Mili -/- E16.5 fetal testis. (b) Quantification of LINE1 and IAP associated pirnas in WT and Mili -/- E16.5 fetal testis. (c) pirnas from WT and Mili -/- E16.5 fetal testis mapped to LINE1 and IAP consensus sequence allowing three mismatches. Positive (red) and negative (blue) values indicate sense and antisense pirnas, respectively. (d)
4 Ping-pong analysis of LINE1 and IAP associated pirnas in WT and Mili -/- E16.5 fetal testis. The relative frequency of the distance between 5 ends of complementary pirnas for LINE1 (right) and IAP (left) is presented. (e) Percentage of LINE1 and IAP associated pirnas in WT and Mili -/- E16.5 fetal testis with a U at the first position (1U) without an A at the position 10 and an A at position 10 (10A) without a U at position 1 is shown. (f) pirnas from WT and Mili -/- E16.5 fetal testis mapped to loci whose methylation is dependent upon MIWI2 and MILI, respectively. Positive (red) and negative (blue) values indicate sense and antisense pirnas, respectively. (g) Quantification of all or uniquely mapping pirna densities across MILI and MIWI2-dependent loci is presented. Significance in e and g was assessed using BootstRatio algorithm, * indicates a p value of < 0.05, ** indicates a p value of < 0.005, *** indicates a p value of < Graphs in a, c, d, f show averages of values obtained from two animals. Individual data points in b, e, g show values obtained from two animals.
5 Supplementary Figure 4 MIWI2 retains partial nuclear localization in the absence of MILI. (a & c) Immunofluorescence with anti-miwi2 antibody (a) and anti-ha antibody (c) on fetal testis of indicated genotypes with a seminiferous tubule shown. (b & d) Quantification of anti-miwi2 (b) and anti-ha (d) fluorescence signal in the nucleus of indicated genotypes. 150 gonocytes from three animals, 100 and 97 gonocytes from two animals were analyzed in panel b from E16.5 Miwi2 -/-, Miwi2 +/- and Mili -/- fetal testes, respectively. 104, 104 and 103 gonocytes from two animals were analyzed in panel d from E16.5 Mili +/-, Mili +/- ; Miwi2 HA/+ and Mili -/- ; Miwi2 HA/+ fetal testes, respectively. Graphs show mean and s.d.
6 Supplementary Figure 5 Generation and validation of the Miwi2 HA allele. (a) A targeting strategy for the generation of Miwi2 HA allele by CRISPR-Cas9 gene editing technology is presented. (b) Chromatogram showing HA-tag and BamHI sequence insertion at the site of starting ATG codon in Miwi2 locus. (c) Validation strategy of Miwi2 HA allele by PCR followed by BamHI restriction is shown. Forward (Fw) and reverse (Rv) primers flanking starting ATG codon amplify 207 bp fragment in presence of Miwi2 + allele and 240 bp fragment in presence of Miwi2 HA allele. When 240 bp fragment is digested with BamHI, 87 bp and 153 bp size fragments are generated (upper panel). PCR amplicon digestion with BamHI is shown as described above (lower panel). (d) Normal testicular weight of adult Miwi2 HA/+ and Miwi2 HA/HA mice is shown. Graph shows mean and s.d. Individual data points are presented.
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