1. Volume 25, Issue 2, Pages 350-356 (October 2018)
Relationship between PIWIL4-Mediated H3K4me2 Demethylation and piRNA- Dependent DNA Methylation 
Ippei Nagamori, Hisato Kobayashi, Toru Nishimura, Reina Yamagishi, Jun Katahira, Satomi Kuramochi-Miyagawa, Tomohiro Kono, Toru Nakano 
Cell Reports 
Volume 25, Issue 2, Pages (October 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 350-356DOI: (10.1016/j.celrep.2018.09.017)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 1
Relationship between piRNA-Dependent DNA Methylation and Histone Modifications in Gonocytes
(A) DNA methylation of control and PIWIL2-null spermatocytes. DNA methylation on the chip probes with more than 20 reads of control and PIWIL2-null spermatocytes is shown. Low-MeC, high-MeC, and piRNA-dependent regions are labeled by purple, yellow, and pink squares, respectively.
(B and C) Relative enrichment of H3K4me2, H3K4me3, and H3K9Ac (B) and H3K9me3 and H3K9me2 (C) in E15.5 gonocytes is shown by boxplots.
(D) Relative enrichment of H3K4me2 in L1Md_A, L1Md_Tf, and IAP regions in E16.0 gonocytes was observed by ChIP-qPCR.
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4. Figure 2 Embryonic H3K4me2 Peaks and piRNA-Dependent DNA Methylation
(A) DNA methylation of control and PIWIL2-null cells. DNA methylation in 500-bp non-overlapping sliding windows with more than 50 methylome reads is shown. Low-MeC, high-MeC, and piRNA-dependent regions are labeled by purple, yellow, and pink squares, respectively.
(B) Percentages of embryonic H3K4me2 peaks neighboring regions in indicated groups are shown.
(C) Percentages of repeat elements at embryonic H3K4me2 peaks neighboring regions in both piRNA-dependent and all regions are indicated by a pie chart.
(D) The number of LINE1 subfamilies occupying indicated groups.
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5. Figure 3
Relationship between H3K4me2 Changes in PIWIL4-Null Spermatogonia and piRNA-Dependent DNA Methylation
(A) Percentages of control and PIWIL4-null H3K4me2 peaks neighboring regions in indicated groups are shown. H3K4me2 peaks in control and PIWIL4-null spermatogonia were defined by the MACS peak caller.
(B) Percentages of H3K4me2-up peaks neighboring regions in indicated groups are shown. H3K4me2-up peaks in PIWIL4-null cells and in control cells were identified using DESeq software (FC > 2, p < 0.05).
(C) The percentages of repeat elements and piRNA-dependent regions with or without H3K4me2 peaks are indicated by a pie chart.
(D) The number of LINE1 subfamilies of indicated groups.
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6. Figure 4
Developmental Changes in H3K4me2 Enrichment and DNA Methylation
(A) Developmental changes in DNA methylation of the whole genome, H3K4me2-up, and piRNA-dependent regions. DNA methylation of E13.5 male PGCs, E16.5 gonocytes, control, and PIWIL2-null spermatocytes in 500-bp non-overlapping sliding windows with more than 50 methylome reads is shown in bean plots.
(B and C) Map viewer images of H3K4me2 enrichment from E16 gonocytes, control, and PIWIL4-null spermatogonia. DNA methylation of E13.5 male PGCs, E16.5 gonocytes, control, and PIWIL2-null spermatocytes in L1Md_A (B) and Rasgrf1 (C) are shown.
(D) Flag-tagged KDMs or PA-tagged PIWIL4-expressing vectors were transfected into 293T cells by polyethylenimine (PEI). Two days after transfection, immunoprecipitation of Flag or PA antibody was performed, followed by western blot using indicated antibodies.
(E) Frozen section of E16.5 testis fixed by 2% PFA. KDM1A or KDM5B and PIWIL4 proteins were visualized by Alexa 488- and Alexa 568-conjugated secondary antibodies, respectively. DNA was counterstained by DAPI after three washes. Images were obtained by BZ-X700 (Keyence). Scale bar: 20 μm.
Cell Reports  , DOI: ( /j.celrep )
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