DEV014340 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Table S2 -

  Adobe PDF

• Supplemental Table S3 -

  Adobe PDF

• Supplemental Table S4 -

  Adobe PDF

• Supplemental Workbook1 -

  Microsoft Excel

• Supplemental Workbook2 -

  Microsoft Excel

• Supplemental Figure S1 -

  Fig. S1. (A) Western blot analysis of total cell lysates (3-fold dilution each) from wild-type, Ring1B-KO (constitutive) and Ring1A-KO ES cells with Ring1A, Ring1B and lamin B antibodies. Lamin B and CBB stainings were used as loading controls. (B) Changes in gene expression levels for Ring1A in wild-type and Ring1B-KO ES cells were determined by real-time PCR, normalized to an Actb control and depicted as fold changes relative to the wild-type ES cells. Error bars are based on the s.d. derived from triplicate PCR reactions.

• Supplemental Figure S2 -

  Fig. S2. Scatter plot of fold expression changes for respective probes in Ring1A/B-dKO and Ring1B-KO ES cells. Fold expression changes for respective probes in Ring1A/B-dKO (day 4; x-axis) and Ring1B-KO (constitutive; y-axis) ES cells determined against the parental cells or wild-type are plotted on a scatter diagram (red dots) and the correlation calculated according to Pearson. N, total number of probes dotted. Variance for Ring1A/B-dKO and Ring1B-KO are indicated as Var(X) and Var(Y), respectively. r, Pearson’s correlation coefficient. Dotted blue lines indicate the first and second feature vectors derived through principal component analysis that correspond to the rotation angle of the distribution. The distribution of red dots is approximated by dotted green ellipses.

• Supplemental Figure S3 -

  Fig. S3. Western blot analysis of changes in protein levels after OHT treatment in Ring1A^−/−;Ring1B^fl/fl;Rosa26::CreERT2 ES cells. Shown are the changes in protein levels for Ring1B, Oct3/4, PRC2 components (Ezh2, Suz12 and Eed), trimethylated histone H3 lysine 27 (H3K27me3), trimethylated histone H3 lysine 4 (H3K4me3) and acetylated histone H3 (H3Ac) 2 days and 4 days after OHT treatment in Ring1A^−/−;Ring1B^fl/fl;Rosa26::CreERT2 ES cells. OHT was absent from (−) or present in (+) the ES cell culture medium. CBB staining for histones was used as a loading control.

• Supplemental Figure S4 -

  Fig. S4. Changes in expression of genes, previously shown to be bound by OCT4, SOX2 and/or NANOG in human ES cells, in Ring1A/B-dKO ES cells. Genes expressed in Ring1A/B-dKO ES cells 2-fold more than the control were designated as ‘derepressed’ genes, whereas those with expression levels below this threshold were designated ‘not derepressed’. Genes bound by OCT4, SOX2 and/or NANOG were assigned based on a previous report (Boyer et al., 2005). We next assigned the mouse genes to human orthologous genes using human and mouse orthology, using the Mouse Genome Database (Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, ME; http://www.informatics.jax.org/mgihome/other/citation.shtml, February, 2007). ‘Active’ and ‘inactive’ genes in human ES cells were defined according to the previous report (Boyer et al., 2005). We found that orthologous genes bound by OCT4, SOX2 and/or NANOG and stably repressed in human ES cells were significantly derepressed in Ring1A/B-dKO ES cells, as compared with those genes in the previous study for which there was no significant binding of OCT4, SOX2 or NANOG (Boyer et al., 2005).

• Supplemental Figure S5 -

  Fig. S5. ES cells expressing Gata6-GR fusion protein (G6GR, −Dex) undergo differentiation directed to primitive endoderm lineages after addition of dexamethasone (+Dex).