SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTRY INFORMTION DOI:.38/ncb Kdmb locus kb Long isoform Short isoform Long isoform Jmj XX PHD F-box LRR Short isoform XX PHD F-box LRR Target Vector 3xFlag Left H Neo Right H TK LoxP LoxP Kdmb Locus TTGTG Exon4 Kdmb Flag knock-in locus (LoxP) Kdmb Flag knock-in locus (LoxP) F 3xFlag Exon4 LoxP F 3xFlag Exon4 LoxP F R Neo LoxP R Right H Right H R D Marker wt (F/R) Targeted (F/R) Marker efore re(f/r) fter re (F/R) E Negative clone Postive clone loxp DPI loxp F ontrol α-tubulin Figure S Establishment of mes line with a Flag-tag knocked into the Kdmb locus. Diagrammatic illustration of the genomic structure of mouse Kdmb locus. The boxes represent exons. Diagrammatic presentation of the long isoform and short isoform of Kdmb protein with their functional domains. Schematic diagram illustrates the strategy for generating Kdmb Flag-tag knock-in mes line. The targeting vector, wild-type Kdmb locus, targeted Kdmb locus and targeted Kdmb locus with deleted selection marker as well as the PR primers for genotyping are indicated (H: homologous arm; Neo: neomycin phosphotransferase; TK: thymidine kinase). Shown are the genotyping results of Kdmb Flag-tag knock-in mes clones. The genotyping PR primer sets are labeled as F, F, R and R. Immunostaining with Flag antibody revealed nuclear localization of endogenous Kdmb-Flag fusion protein in the FLG-tag knock-in mes line. bars = μm. Western blot analysis demonstrates that Flag antibody detected the endogenous Kdmb-Flag protein in the Flag-tag knock-in mes line, while the signal is greatly reduced in the same mes line with Kdmb knockdown. 3 Macmillan Publishers Limited. ll rights reserved.

2 Relative expression Relative expression SUPPLEMENTRY INFORMTION 3.5 Kdmb SF. Kdmb SF MEF Min6 EG ES NP D D3 D6 D9 Figure S Oct4 and Sox bind to the promoter of Kdmb. Relative expression levels of Kdmb short isoform in embryonic stem cells (ES), embryonic germ cells (EGS), neural stem cells (NS), murine embryonic fibroblasts (MEF), myoblasts and pancreatic β cell line (Min6) measured by RT-qPR. Values represent means ± standard deviation from three biological replicates. Relative expression levels of Kdmb short isoform at different days of embryoid body differentiation measured by RT-qPR and normalized with Gapdh. The mrn level of mess (D) is arbitrarily set as. Values represent means ± standard deviation from three biological replicates. 3 Macmillan Publishers Limited. ll rights reserved.

3 ell numbers (x ) SUPPLEMENTRY INFORMTION α-oct4 α-sox DPI DPI α-nanog DPI D (Days) E % of colony formation 8 6 Figure S3 Depletion of Kdmb in mess does not affect expression of pluripotent genes, cellular proliferation and clonogenicity. (-) Immunostaining of Oct4 (), Sox (), and Nanog () in control, and Kdmb rescued mess. bars = μm. (D) Growth curve of control, Kdmb knockdown and Kdmb rescued mess. Values represent means ± standard deviation from three biological replicates. (E) olony formation capacity of control, Kdmb knockdown and Kdmb rescued mess. Values represent means ± standard deviation from three biological replicates Macmillan Publishers Limited. ll rights reserved.

4 Expression Expression Expression Expression Expression SUPPLEMENTRY INFORMTION mock wild-type mut Jmj del XX wild-type mut Jmj mut XX mut PhD del Fbox del LRR short isoform α-tubulin Kdmb Mesoderm Ectoderm T Mix Sox Fgf5 Pluripotent Genes Polycomb Oct4 Nanog Sox mi Ringb Ezh ontrol +Wild-type +Jmj mut. +XX del. +XX mut. +PHD mut. +Fbox del. +LRR del. +SF Figure S4 Knockdown of Kdmb does not affect expression of mesoderm, ectoderm, pluripotent and Polycomb genes. Relative expression levels of Kdmb in the control, Kdmb knockdown as well as the wild-type and various mutants rescued mess measured by RT-qPR. The mrn level of Kdmb in control knockdown mess is arbitrarily set as. Values represent means ± standard deviation from three biological replicates. Western blot analyses demonstrate the expression of the exogenous wild-type, various mutants and short isoform of Kdmb. Tubulin serves as a loading control. RT-qPR analysis demonstrates none of the representative mesoderm, ectoderm, pluripotent and Polycomb genes are altered in the Kdmb knockdown or various mutant rescued mess. The mrn level of control knockdown mess is arbitrarily set as. Values represent means ± standard deviation from three biological replicates Macmillan Publishers Limited. ll rights reserved.

5 SUPPLEMENTRY INFORMTION Kdmb-Flag wild-type del cxxc wild-type del cxxc DPI Wild-type MEF Dnmt/p53 DKO MEF Figure S5 Kdmb binds to unmethylated GIs in mess. Representative immunostaining images show the localization of wild-type and XX-ZF deletion Kdmb mutant in the wild-type and the Dnmt/p53 double knockout (DKO) MEF. Note that the wild-type Kdmb, but not the XX-ZF deletion mutant, is localized to DPI heavy foci in the Dnmt/p53 MEFs. bars = 5μm Macmillan Publishers Limited. ll rights reserved.

6 SUPPLEMENTRY INFORMTION oding region 3kb kb TSS TES kb 3kb Genomic ackground Kdmb occupancy. Distal promoter (-3 kb). Proximal promoter (< kb) 3. Intragenic region -5 UTR -coding region -intron.5%.3% % 9.8% 43% 54.8%.3% % 39.4%.% 6.% 6.7% -3 UTR 4. Immediate downstream (< kb).3% %.8%.9% 5. Distal downstream (-3 kb).5%.5% 6. Distal intergenic (>3 kb) 5% 3.7% Figure S6 The genomic distribution of Kdmb occupancy. Pie chart shows the genomic distribution of Kdmb occupancy in mess. The percentage of Kdmb occupancy at each genomic region is listed Macmillan Publishers Limited. ll rights reserved.

7 % of Input % of Input % of Input % of Input % of Input SUPPLEMENTRY INFORMTION α Flag (Kdmb SF) α-ringb % Input 5% IgG IP 5% Flag IP Normalized read density (RPM) (ntibody - IgG) RINGb occupancy Normalized read density (RPM) (ntibody - IgG) 5 5 EZH occupancy Distance to the peak of Distance to the peak of Kdmb binding center (kb) Kdmb binding center (kb) Gata6 Sox7 Eomes Ezh ontrol ESs Ezh ontrol ESs Ezh ontrol ESs Ezh ESs Ezh ESs Ezh ESs Ezh hip-qpr IgG α-ezh IgG α-ezh ontrol cells cells.5.5 Ezh hip-qpr IgG ontrol α-ezh cells IgG α-ezh cells Ezh hip-qpr IgG ontrol α-ezh cells IgG α-ezh cells D E ontrol Ringb KD.5 Gata6.6 Eomes.5 α-ringb (Kdmb) α-tubulin IgG IgG ontrol cells Ringb KD cells Figure S7 Kdmb is required for the recruitment of PR to the GIs of target genes. o-immunoprecipitation and Western blot analyses show the short isoform of Kdmb fails to co-immunoprecipitate with Ringb. Genome-wide occupancy of Ringb and Ezh at the Kdmb-bound genes that do not have changed gene expression upon Kdmb knockdown in control (black) and Kdmb knockdown (red) mess. verage signal within 5 kb genomic regions flanking the center of Kdmb peaks is shown. Shown on the top panels are the results of Ezh at three representative genes Gata6, Sox7, and Eomes in control and Kdmb knockdown mess. Shown on the bottom panels is the relative enrichment of Ezh occupancy at the same sites. The enrichment of occupancy was measured by hip-qpr. Values represent means ± standard deviation from three biological replicates. Western blot analyses show that Ringb and Kdmb protein levels at 48 hours after mess are transduced with control or lentiviarl Ringb-shRN vector. Tubulin serves as a loading control. Shown is the relative enrichment of Kdmb occupancy at two representative genes Gata6, and Eomes in control and Ringb knockdown mess. The enrichment of occupancy was measured by hip-qpr. Values represent means ± standard deviation from three biological replicates Macmillan Publishers Limited. ll rights reserved.

8 SUPPLEMENTRY INFORMTION Kdmb LF Kdmb SF D E Figure S8. Scanned images of immunoblots. The boxed regions indicate the bands shown in the figures. () Fig. ; () Fig. F; () Fig. 5; (D) Fig. 5D; (E) Fig Macmillan Publishers Limited. ll rights reserved.