A region 627 794 SETDB 1 binding m
A region 627 -794 (SETDB 1 binding) m. ATF 7 IP(606 -705): h. ATF 7 IP(572 -671): estimated α-helix region 627 -694 a. a. of mouse ATF 7 IP B region 1190 -1306 (FN III domain) m. ATF 7 IP(1190 -1306): h. ATF 7 IP(1160 -1270): m. ATF 7 IP: h. ATF 7 IP: Figure S 1 estimated α-helix
A Atf 7 ip KO Day 5 B SETDB 1 +3 x. F-ATF 7 IP 100 d. FNIII % intensity (nucleus/total) WT SETDB 1 80 60 40 20 + d. F N W III T 0 K A A tf 7 ip O K + O FLAG C DAPI ATF 7 IP % intensity (nucleus/total) 100 SETDB 1 +FLAG 80 60 40 20 d. F N W + + O K tf 7 ip A Figure S 2 O K tf 7 ip 10µm A SETDB 1 +DAPI III T 0
Identified protein network in the top 20 candidate proteins Figure S 3
FAM 1 B FAM 2 182 …IQIANVTTLETGVSSVSDGQLESTDGRDMNLMITHVTSL… 220 1 180 350 780 Mym-domain (k. Da) IP: α-V 5 ZMYM 2 (mouse) : V 5 -m. ZMYM 2 + + + : 3 x. F-m. ATF 7 IP WT FL Δ 1 Δ 2 Δ 3 Δ 4 A 1376 α-FLAG 250250 - α-V 5 150100 - LSD 1 -HDAC Input FL (2 -1376): Δ 1 (2 -1249): α-FLAG 250250150 - α-V 5 100 - Δ 2 (2 -1098): 1 Δ 3 (2 -999) : Δ 5 (2 -603) : (k. Da) IP: α-V 5 Δ 6 (2 -443) : Δ 7 (2 -350) : Δ 8 (2 -211) : 3 4 5 6 : V 5 -m. ZMYM 2 + + + : 3 x. F-m. ATF 7 IP WT α-FLAG 250250150 - α-V 5 100 - ΔN 1 (181 -1376) : Input ΔN 2 (190 -1376) : ΔN 3 (694 -1376) : : V 5 -m. ZMYM 2 + + + : 3 x. F-m. ATF 7 IP WT α-FLAG IP: α-V 5 25010075 - α-V 5 5037 - α-FLAG Input 25010075 - α-V 5 50371 2 3 4 150 - α-V 5 1 Δ 4 Δ 5 Δ 6 Δ 7 Δ 8 (k. Da) α-FLAG 250250 - 100 - ΔN 4 (781 -1376) : D Cells: HEK 293 T FL ΔN 1 ΔN 2 ΔN 3 ΔN 4 C Δ 4 (2 -811) : 2 5 6 Cells: HEK 293 T Figure S 4 2 3 4 5 6 Cells: HEK 293 T
α-V 5 75 - α-FLAG 250 - α-V 5 751 2 3 C WT FAM Mut (k. Da) Cells: HEK 293 T : V 5 -m. ZFP 518 A + + : 3 x. F-m. ATF 7 IP WT α-FLAG 250250 - α-V 5 1 2 3 4 Figure S 5 Input IP: α-V 5 α-FLAG 250 - WT FAM Mut ΔFAM : V 5 -m. MGA C-ter + + + : 3 x. F-m. ATF 7 IP WT B IP: α-V 5 (k. Da) Cells: HEK 293 T Input IP: α-V 5 WT FAM Mut A (k. Da) Cells: HEK 293 T : V 5 -m. ZMYM 4 + + + : 3 x. F-m. ATF 7 IP WT α-FLAG 250250 - α-V 5 1 2 3
A B C WT Atf 7 ip KO Zmym 2 KO + WT 25 * ** Relative to WT NS 15 ** NS 20 ** ** NS ** ** 10 5 0 Fkbp 6 Mael 1700019 A 02 Rik Figure S 6 Rec 114
A B RNA-seq sample Repliacte Description WT m. ESCs 2 two replicates from one WT m. ES cell line Atf 7 ip KO 2 two cell lines Atf 7 ip KO + WT 4 two replicates from each cell line Atf 7 ip KO + d. FNIII 4 two replicates from each cell line Zmym 2 KO 2 two cell lines Ch. IP-seq sample Repliacte Description Flag-ATF 7 IP 2 two replicates from one cell line Flag-ATF 7 IP d. FNIII 2 two replicates from one cell line Correlation between WT replicate Correlation between Atf 7 ip KO clones Pearson’s r = 0. 99 WT rep 1 Atf 7 ip KO (TT 2 -5) Correlation between WT ATF 7 IP rescue Correlation between d. FNIII ATF 7 IP rescue Atf 7 ip KO (TT 2 -12) + WT ATF 7 IP Atf 7 ip KO (TT 2 -12) + d. FNIII ATF 7 IP Pearson’s r = 0. 99 Atf 7 ip KO (TT 2 -5) + WT ATF 7 IP C Pearson’s r = 0. 99 Atf 7 ip KO (TT 2 -5) + d. FNIII ATF 7 IP Gene expression Log 2 (RPM+1) WT Atf 7 ip KO (TT 2 -5) Zmym 2 KO WT rescue (TT 2 -5) WT Atf 7 ip KO (TT 2 -12) Pearson’s r = 0. 99 Zmym 2 KO #2 WT rep 2 Atf 7 ip KO (TT 2 -12) Pearson’s r = 0. 98 Correlation between Zmym 2 KO clones d. FNIII rescue (TT 2 -5) WT rescue (TT 2 -12) d. FNIII rescue (TT 2 -12) Figure S 7 Zmym 2 KO #1
Zmym 2 KO 2 Zmym 2 KO 1 Atf 7 ip KO (87) Atf 7 ip KO-TT 2 -12 Atf 7 ip KO-TT 2 -5 C A Atf 7 ip KO /Zmym 2 KO 52 Atf 7 ip KO 4 KO TT 2 -12 +d. FNIII KO TT 2 -5 KO TT 2 -12 KO TT 2 -5 D KO TT 2 -12 +WT Atf 7 ip KO /Zmym 2 KO Atf 7 ip KO Log 2(FC) KO TT 2 -12 -4 KO TT 2 -5 Zmym 2 KO 2 Zmym 2 KO 1 Atf 7 ip KO-TT 2 -12 B Atf 7 ip KO-TT 2 -5 Zmym 2 KO (178) Zmym 2 KO -4 Log 2(FC) 4 -4 Atf 7 ip KO/Zmym 2 KO u u u Meiotic cell cycle (Fkbp 6/Hormad 1/Aurkc/Mael/Stra 8/Sycp 1/Tdrd 12/Tex 15) Spermatogenesis (Fkbp 6/Hormad 1/Aurkc/Mael/Morc 1/Rpl 39 l/Stra 8/Sycp 1/Tdrd 12/Tdrd 15/Uba 1 y) DNA methylation involved in gamete generation (Fkbp 6/Mael/Morc 1/Tdrd 12) Synapsis (Hormad 1/Mael/Stra 8/Sycp 1/Tex 15) Spermatid development (Xlr 3 a/Xlr 3 b/Xlr 5 a/Xlr 5 b/Xlr 5 c) Figure S 8 Log 2(FC) 4
A WT: rep 1+2 6537 d. FNIII: 3376 3177/3376=94. 1% B Ch. IP Flag-ATF 7 IP d. FNIII WT m. ESCs RNA Atf 7 ip KO m. ESCs Atf 7 ip KO + WT Atf 7 ip KO + d. FNIII Dazl Ch. IP Flag-ATF 7 IP d. FNIII WT m. ESCs RNA Atf 7 ip KO m. ESCs Atf 7 ip KO + WT Atf 7 ip KO + d. FNIII Fkbp 6 Figure S 9
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