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Supplemental Information Posfai Et Al., Polycomb Function During Supplemental Information Posfai et al., Polycomb function during oogenesis is required for mouse embryonic development Supplemental Figures and Legends Figure S1. Ring1 and Rnf2 gene deletion strategy during gametogenesis and transcript and protein levels of PRC1 components in wild- type and mutant growing oocytes and early embryos . Figure S2. Reconstitution of endogenous-like levels of PRC1 complex in Ring1m-z+/Rnf2m-z+ early zyotes does not alleviate the developmental arrest at the two-cell stage Figure S3. Ring1 and Rnf2 gene deletion during gametogenesis results in delayed meiotic maturation of oocytes, delayed early embryonic development and arrest before entry into the second cleavage division Figure S4. DNA replication analysis, γ-H2AX patterns and checkpoint activation in control and Ring1m-z+/Rnf2m-z+ embryos Figure S5. Transcriptional shut-down occurs in Ring1/Rnf2 dm GV oocytes Figure S6. Majority of genes misregulated in Ring1/Rnf2 dm GV oocytes are normally expressed in Ring1- or Rnf2 single mutant oocytes Figure S7. Transcripts of developmental regulators de-repressed in Ring1/Rnf2 dm oocytes are only translated after fertilization Figure S8. Ring1/Rnf2 expression during oogenesis defines maternal cytoplasmic and nuclear contributions required for embryonic development. Supplemental Tables Table S1. Gene Ontology (GO) categories of genes up- or down- regulated in Ring1/Rnf2 dm GV oocytes. Table S2. Comparison of Ring1/Rnf2 dm GV oocyte expression profiling data to expression in Rnf2/Eed double mutant mouse ESCs (Leeb et al., 2010). Table S3. Comparison of Ring1/Rnf2 dm GV oocyte expression profiling data to Rnf2 binding profile in wild-type mouse ESCs (Endoh et al., 2008). Table S4. Gene Ontology (GO) categories of genes up- or down- regulated in Ring1/Rnf2 dm GV oocytes, classified according to expression levels of genes during subsequent stages of normal oogenesis. Supplemental Experimental Procedures Mice Antibodies Quantitative real-time RT-PCR primers Comparison to published data sets Comparison to expression patterns during oocyte development and early embryogenesis Defining expression states Defining expression profiles Comparison to expression data of PRC1 or PRC1/PRC2 deficient mouse ESCs and genome wide Rnf2 binding data in mouse ESCs Comparison to H3K27me3 Chip-seq data of mouse ESCs, MEFs and human spermatozoa Statistical analyses A Wild-type spermato spermatid elongating spermatozoon -gonia spermatid +/+ x +/+ early zygote late zygote 2-cell embryo 8-cell blastocyst primordial primary secondary small large GV M-II oocyte oocyte oocyte antral antral oocyte oocyt oocyte oocyte Zygotic transcription Maternal contribution Pan et al., 2005 Zeng et al., 2004 and Zeng et al., 2005 B Conditional mutants spermato spermatid elongating -gonia spermatid spermatozoon F/F Prm1-cre x Zp3-cre Prm1-cre F/F Zp3-cre early zygote late zygote 2-cell embryo 8-cell blastocyst primordial primary secondary small large GV M-II oocyte oocyte oocyte antral antral oocyte oocyt oocyte oocyte Zygotic transcription Maternal contribution C Ring1 Phc1 Rnf2 Phc2 Cbx2 Phc3 relative expression levels primary secondary GV zygote 2-cell Bmi1 oocyte oocyte oocyte wild-type Rnf2 mutant Ring1 mutant Ring1/Rnf2 dm primary secondary GV zygote 2-cell oocyte oocyte oocyte D DAPI Ring1 DAPI Rnf2 DAPI Cbx2 DAPI Bmi1 DAPI Rybp wild-type Rnf2 mutant Ring1 mutant Growing oocyte dm Ring1/Rnf2 E DAPI Ring1DAPI Rnf2 DAPICbx2 DAPI Bmi1 wild-type m-z+ Rnf2 m-z+ 2-cell embryo Ring1 / m-z+ m-z+ Rnf2 Ring1 F Rnf2 Ring1 Ring1/Rnf2 wild-type mutant mutant dm growing mRNA/protein mRNA/protein mRNA/protein mRNA/protein oocyte Ring1 + + + ++ - - - - Rnf2 + + - - + + - - Cbx2 + + + + + + + - Bmi1 + + + + + + + - Ring1m-z+/ wild-type Rnf2m-z+ Ring1m-z+ Rnf2m-z+ 2-cell mRNA/protein mRNA/protein mRNA/protein mRNA/protein embryo Ring1 - - - - - - - - Rnf2 + + - - + + - - Cbx2 + + + - + + + - Bmi1 + + + - + + + - Developmental potential + + + - (beyond the 2-cell stage) Supplemental figure 1 Ring1 and Rnf2 gene deletion strategy during gametogenesis and transcript and protein levels of PRC1 components in wild-type and mutant growing oocytes and early embryos (A) Schematic diagram illustrating different stages of gametogenesis and early embryonic development. Com- parative expression analyses described in Figure 5C and Figure S6D were based on genome-wide transcriptome analyses in wild-type oocytes at five subsequent stages of follicle development (from primordial to large antral follicle stage) (Pan et al., 2005) and in wild-type GV oocytes and early embryos (late zygotes, 2-cell, 8-cell and blastocyst embryos) (Zeng et al., 2004; Zeng and Schultz, 2005). (B) Conditional genetic strategy to generate embryos maternally and zygotically deficient for Rnf2 function. Zp3-cre drives Cre recombinase expression during oogenesis from the primary follicle stage onwards, resulting in loss of Rnf2 expression during the transcriptionally active growth phase of oogenesis. Prm1-cre drives Cre recombinase expression in late round spermatids and during the elongation phase of spermiogenesis during which transcription is largely shut-down in a genome-wide manner. Deletion of Ring1 and Rnf2 in maturing sper- matids did not impair embryonic or postnatal development of double heterozygous offspring (data not shown). The lack of a paternal affect phenotype may be due to the conditional deletion of Rnf2 relatively late during sper- miogenesis, a developmental phase of minor transcriptional activity as compared to Zp3-cre mediated deletion in actively transcribing growing oocytes. (C) Quantitative real-time PCR analysis of gene transcripts of Ring1, Rnf2 and of other PRC1 members Cbx2, Bmi1, Phc1, Phc2 and Phc3 in growing oocytes (from primary and secondary follicles), GV oocytes, zygotes and two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. Transcript levels were normalized to LnmB1 control. Error bars indicate standard deviation based on 2-3 biological replicates. (D) IF staining for Ring1, Rnf2, Cbx2, Bmi1 and Rybp in wild-type, Rnf2 mutant (Rnf2F/F; Zp3-cre), Ring1 mutant (Ring1-/-) and Ring1/Rnf2 dm (Ring1-/-/Rnf2F/F; Zp3-cre) growing oocytes (from secondary follicles) surrounded by follicle cells in ovary sections from 12-14 day old mice. Ring1 protein level is increased in Rnf2 mutant oocytes. Due to constitutive deletion granulosa cells lack Ring1 protein in Ring1 mutant ovaries. >20 follicles were analyzed from 6 mice for each IF staining. Scale bar = 10 μm. (E) IF staining for Ring1, Rnf2, Cbx2 and Bmi1 in two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. Scale bar = 20 μm. (F) Summary of mRNA and protein expression of PRC1 components in growing oocytes and two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. A m-z+ m-z+ C Ring1 /Rnf2 control control Rnf2 mRNA water water DAPI Rnf2 merge injected injected injected O 2 H DAPI Ring1m+z+; Rnf2m-z+ DAPI Rnf2 merge Bmi1 O 2 H B Rnf2 50 ng Rnf2 mRNA 10 ng relative mRNA expression levels Rnf2 mRNA H2OH2O 50ng 10ng1ng 0.1ng m+z+ m-z+ control Ring1 ; Rnf2 1 ng D Rnf2 mRNA Rnf2 0.1 ng Rnf2 mRNA relative protein expression levels H2O H2O 50ng 10ng 1ng 0.1ng (9) (4) (5) (8) (8) (5) control Ring1m+z+; Rnf2m-z+ E 50 ng Rnf2 mRNA injected 2 ng Rnf2 mRNA injected control Ring1m-z+; Rnf2m-z+ control Ring1m-z+; Rnf2m-z+ 100 100 80 80 60 60 40 40 % of embryos 20 % of embryos 20 0 0 H2O Rnf2 mRNA H2O Rnf2 mRNA H2O Rnf2 mRNA H2O Rnf2 mRNA (45) (30) (26) (30) (35) (27) (21) (30) fragmented 2-cell 4-cell morula zygote 3-cell 8-cell blastocyst Supplemental figure 2 Reconstitution of endogenous-like levels of PRC1 complex in Ring1m-z+/Rnf2m-z+ early zyotes does not alleviate the developmental arrest at the two-cell stage (A) Microinjection of myc-tagged Rnf2 mRNA into Ring1m-z+/Rnf2m-z+ zygotes leads to de-novo PRC1 com- plex formation. IF analyses of control and Ring1m-z+/Rnf2m-z+ zygotes microinjected with water or with myc- tagged Rnf2 mRNA. Embryos were stained with anti-Bmi1 antibodies to visualize reconstitution of chromatin bound PRC1. Scale bar = 20 μm. (B) Rnf2 mRNA levels measured by quantitative real-time PCR in water injected control zygotes and Ring1m- z+/Rnf2m-z+ zygotes microinjected with 0 (water), 50, 10, 1, 0.1 ng of myc-tagged Rnf2 mRNA. Early IVF zygotes were injected 3-5 hpf and harvested 12 hpf. Transcript levels were normalized to LnmB1 control. Error bars indicate standard deviation based on 2-3 biological replicates. (C) IF staining with anti-Rnf2 antibody of water injected control zygotes and Ring1m-z+/Rnf2m-z+ zygotes microinjected with 0 (water), 50, 10, 1, 0.1 ng of myc-tagged Rnf2 mRNA. Early IVF zygotes were injected 3-5 hpf and harvested 12 hpf. Scale bar = 20 μm. (D) Quantification of Rnf2 protein levels by measuring total fluorescent signal (Y axis) of IF staining with anti- Rnf2 antibody in panel (B) embryos. Total fluorescent signal was measured from both PN together. Error bars indicate standard deviation based on the number of embryos analyzed (indicated in brackets). (E) Microinjection of 50 (left panel) or 2 (right panel) ng of myc-tagged Rnf2 mRNA into early zyotes does not alleviate the developmental arrest at the two-cell stage. Diagram showing developmental potential of control and Ring1m-z+/Rnf2m-z+ embryos, microinjected at the early zygote stage with water or Rnf2 mRNA. Developmen- tal potential was scored at embryonic day 3.5. A 21 day ovary control Ring1-/-; Rnf2F/F; Zp3-cre B C 100 80 2.5-3.5
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