The SETD7 knock-down affects the cell cycle profile of differentiating cells.

<p>(A) Cell cycle profile of undifferentiated and after 15 days of <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7). (B) Mean of the percentage of cells in each phase of the cell cycle in three independent differentiation experiments. *Differences in the percentage of cells in S-phase between shSETD7 and shSCR in differentiated cells was found significant at a p-value<0.05.</p

Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.

<p>Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.</p

The SETD7 knock-down causes defects in the silencing of pluripotency genes.

<p>(A) Western blot showing the levels of OCT4 at different days during the in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (B) Immunolocalization of SOX2 (green) and OCT4 (red) expression at day 4 and day 15 of in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (C) Quantification of the percentage of embryoid bodies negative or positive for OCT4 staining at day 4 and day 7 of differentiation of cells treated with vehicle (DMSO), 1μM or 5μM PFI-2. (D) Immunolocalization of OCT4 (red) in embryoid bodies at day 4 and day 7 of in vitro differentiation of ES[4] treated with vehicle (DMSO) or 5μM PFI-2.</p

SETD7 interacting proteins identified by mass spectrometry.

<p>SETD7 interacting proteins identified by mass spectrometry.</p

SETD7 is expressed at very low levels in pluripotent human cells and induced during differentiation.

<p>(A) Average rank of the top 700 most differentially expressed genes between pluripotent (iPSCs or ESCs) and fibroblasts, including those upregulated in pluripotent cells (left panel) and upregulated in fibroblasts (right panel). (B) <i>SETD7</i> mRNA levels in human ESCs grown under self-renewal conditions (UndES[4]), <i>in vitro</i> differentiated human ESCs (DifES[4]), human fibroblasts (HFF), two lines of human keratinocytes (HEK1 and HEK2) and two lines of iPSCs generated from keratinocytes ([H]KiPS4F and KiPS4F1). Mean and standard deviation of three technical replicates is shown. Induction of SETD7 mRNA levels during ES[4] differentiation was confirmed in more than four independent differentiation experiments. (C) Western blot showing SETD7 protein levels in pluripotent and somatic cells. Loading control beta actin (ACTB) is also shown. (D) Western blot showing protein levels of SETD7, AFP, OCT4 and SOX2 in under self-renewing conditions and <i>in vitro</i> differentiated human ESCs. Loading control alpha tubulin (TUBA) is also shown. One representative experiment out of three is shown. (E) Genomic visualization of the levels of H3K72me3, H3K4me3, H3K4me2, H3K36me3 and RNA polymerase II (Pol II) in the human embryonic stem cell line H1 around the <i>SETD7</i> gene according to ENCODE. A non-methylated CpG island is depicted in green. (F) Levels of H3K4me2 and H3K27me3 at <i>SETD7</i> gene promoter region (27 bp upstream of the transcription start site) in pluripotent and somatic cells determined by chromatin immunoprecipitation (ChIP) and ploted relative to the input. IgGs wer used as negative control. Bars show the mean and standard deviation of three independent immunoprecipitations.</p

The SETD7 knock-down causes a delay in differentiation.

<p>(A) mRNA levels of pluripotency factors (<i>OCT4</i>, <i>SOX2</i>, <i>NANOG</i>), differentiation genes (<i>HNF4</i> and <i>p21</i>) and <i>SETD7</i> normalized to <i>GAPDH</i> at different days during the <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA (sh28) that targets SETD7 (shSETD7). For pluripotency factors levels were plotted relative to d0 and for differentiation factors levels were plotted relative to day of maximum expression in the shSCR. Mean and standard deviation from three independent differentiation experiments is shown. (B) Gene set enrichment analysis (GSEA) of the global changes in gene expression found between shSETD7 and shSCR at day 8 of differentiation. Enrichment profile, normalized enrichment score (NES), p-value and false discovery rate (FDR) are shown for the significantly enriched gene sets from the Molecular Signatures Database BENPORATH_ES_1 (genes overexpressed in human embryonic stem cells according to 5 or more out of 20 profiling studies), HSIAO_LIVER_SPECIFIC_GENES (Liver selective genes), ESC_J1_UP_LATE.V1_UP (Genes up-regulated during late stages of differentiation of embryoid bodies from J1 embryonic stem cells) and CAHOY_ASTROGLIAL (Genes up-regulated in astroglia cells). (C) Heatmap of the expression of the 400 genes most differentially regulated by shSETD7 KD compared to shSCR and that are upregulated or downregulated more than 16 fold during differentiation determined by microarray analysis.</p

Analysis of SETD7 interacting proteins.

<p>(A) Strategy for the purification of interacting proteins. (B) Levels of endogenous and overexpressed SETD7 in infected HeLa cells infected with pWPI-FLAG (FLAG) or pWPI-FLAG:SETD7 (FLAG:SETD7) determined by western blot. (C) Coomassie blue staining of the immunoprecipitated proteins. Molecular weight markers, anti FLAG-immunoprecipitated proteins from cells transduced with empty vector and cells transduced with FLAG:SETD7 expressing vector are shown.</p