Comparison of epigenetic modifier gene expression across cell lineages.

<p>(<b>A</b>) The expression of 156 modifier genes was measured for 4 differentiated cell types as specified in Fig. 1–3, and transcript levels were all normalized to those of hESC. Three independent assays (#1–#3) were performed for each cell type, and the 12 data sets were represented as heat map. Red color indicates z-scores >0 and yellow color indicates z-scores <0. The data sets were clustered according to their Euclidean distances, as indicated by the dendrogram on top. The individual example genes depicted on the right in black show large differences between liver (huHep, Hep-like) and brain (Ctx, Lu d6). Genes with similarities in non-dividing cells (Ctx, Lu d6, huHep) are shown in purple; those with differences between primary and stem cell-derived cells are shown in red.</p

Synopsis of the regulation of euchromatin associated epigenetic modifier groups in different cell lineages.

<p>Three groups of epigenetic modifiers were selected for a comparison of relative expression levels of Lu d6, Ctx, Hep-like and huHep. Data were obtained, as described in Fig. 1–3. All data are means ± SEM of three independent differentiations. (<b>A, B</b>) Comparison of relative expression levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in Lu d6, Ctx, Hep-like and huHep cells. (<b>C)</b> Genes responsible for euchromatin establishment and maintenance.</p

Comparison of histone modifier sets between LUHMES cells and Cortex samples.

<p>(<b>A</b>) LUHMES cells (Lu0) were differentiated for 6 days (Lu d6) and stained with antibodies specific for NeuN, synaptophysin and TUJ1. Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 100 µm. (<b>B</b>) RT-qPCR data from three independent experiments for dopaminergic and neuronal differentiation markers. Relative gene expression was calculated using day0 as a calibrator and a set of three reference genes (HPRT, RPL13A, GAPDH) (<b>D</b>) Transcript levels of epigenetic modifiers were measured by RT-qPCR in human Cortex (Ctx), LUHMES day6 (Lu d6) and embryonic stem cells (hESC). Data for Ctx and Lu d6 are indicated as relative change compared to hESC (as reference cell). For comparative display, a scatter plot was constructed so that differentially expressed genes that show pos. association (between Ctx and Lu d6) are found in red fields, and those that differed in the sense of regulation fall into blue fields. Values of >10 were set to 10. For quadrant count ratio analysis (QCR) only expression values >2 or <−2 were included. (<b>E</b>) The data measured in D were plotted as heat map, sorted according to rel. Ctx expression levels. Transcripts that were >2-fold higher expressed in tissue than in hESC are marked in red, >2-fold lower expression is marked in blue. The color scale ranges from a fold regulation of −20 (dark blue) to +20 (dark red). Measures of variance and p-values are indicated in the supplemental material, genes not regulated significantly (vs. hESC) are displayed as “n.s.”.</p

Synopsis of the regulation of heterochromatin associated epigenetic modifier groups in different cell lineages.

<p>Three groups of epigenetic modifiers were selected for a comparison of relative expression levels of Lu d6, Ctx, Hep-like and huHep. Data were obtained, as described in Fig. 1–3. All data are means ± SEM of three independent differentiations. (<b>A</b>) Genes responsible for heterochromatin establishment and maintenance. (<b>B, C</b>) Genes that are involved in polycomb complex (PRC1, PRC2) formation.</p

Comparison of epigenetic modifier transcript levels between liver and brain.

<p>(<b>A</b>) Schematic diagram showing sample preparation and analysis of a set of 156 epigenetic modifier genes. (<b>B</b>) Human hepatocytes were stained 24 h after plating with antibodies specific for dipeptidyl peptidase (DPP4) or albumin (ALB). Nuclei were stained with the DNA dye H-33342 (blue). Data are representative for preparations from three different donors. Scale bars: 100 µm. (<b>C</b>) The mRNA was isolated from three preparations of freshly-isolated hepatocytes and analyzed by RT-qPCR for hepatic (ALB, CYP3A, CYP7A1, DPP4, HNF4, MET) and neuronal (TH, DCX, TUBB3) differentiation markers. Gene expression levels are indicated relative to hESC as reference cell line and a set of three reference genes (HPRT, RPL13A, GAPDH) was used for internal calibration. (<b>D</b>) Transcript levels of epigenetic modifiers were measured by RT-qPCR in human cortex (Ctx), liver (huHep) and embryonic stem cells (hESC). Data for Ctx and huHep are indicated as relative change compared to hESC (as reference cell). For comparative display, a scatter plot was constructed so that differentially expressed genes that show pos. association (between Ctx and huHep) are found in red fields, and those that differed in the sense of regulation fall into blue fields. Values of>10 were set to 10. For quadrant count ratio analysis (QCR) only expression values >2 or <−2 were included. (<b>E</b>) The data measured in D were plotted as heat map, sorted according to rel. Ctx expression levels. Transcripts that were >2-fold higher expressed in tissue than in hESC are marked in red, >2-fold lower expression is marked in blue. The color scale ranges from a fold regulation of −20 (dark blue) to +20 (dark red). Measures of variance and p-values are indicated in the supplemental material, genes not regulated significantly (vs. hESC) are displayed as “n.s.”. Specific examples of differential regulation between Ctx and huHep are emphazised by black boxes.</p

Comparison of histone modifier sets between two hepatic cell cultures.

<p>(<b>A</b>) Differentiation scheme of hESC towards Hepatocyte-like cells (Hep-like). (<b>B</b>) Hep-like cells were generated from hESC and stained with antibodies specific for DPP4 and albumin. Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 100 µm. (<b>C</b>) RT-qPCR data from three independent experiments for hepatic (ALB, CYP3A, CYP7A1, DPP4, HNF4, MET) and neuronal (TH, DCX, TUBB3) markers. Relative gene expression was calculated using hESC as a calibrator and a set of three reference genes (HPRT, RPL13A, GAPDH). (<b>D</b>) Transcript levels of epigenetic modifiers were measured by RT-qPCR in human liver (huHep), Hep-like islets (Hep-like) and embryonic stem cells (hESC). Data for huHep and Hep-like are indicated as relative change compared to hESC (as reference cell). For comparative display, a scatter plot was constructed so that differentially expressed genes that show pos. association (between Hep-like and huHep) are found in red fields, and those that differed in the sense of regulation fall into blue fields. Values of >10 were set to 10. For quadrant count ratio analysis (QCR) only expression values >2 or <−2 were included. (<b>E</b>) The data measured in D were plotted as heat map, sorted according to rel. huHep expression levels. Transcripts that were >2-fold higher expressed in tissue than in hESC are marked in red, >2-fold lower expression is marked in blue. The color scale ranges from a fold regulation of −20 (dark blue) to +20 (dark red). Measures of variance and p-values are indicated in the supplemental material, genes not regulated significantly (vs. hESC) are displayed as “n.s.”.</p

Alternative early neural differentiation to neural crest progenitors.

<p>(A) hESC were differentiated towards NCP and stained with antibodies specific for <i>OCT4</i> (no stain observed), <i>PAX6</i> (no stain observed), <i>NESTIN</i> and <i>HNK-1</i>. Cell nuclei were labeled with the DNA dye Hoechst H-33342 (blue). Scale bars: 100 µm. (B) Pairwise comparisons of hESC, NEP or NCP yielded 4277 differentially expressed transcripts. The heat map displays the genes after clustering according to the Pearson's correlation of their expression values across samples. The colors represent Z-scores of the row-wise normalized expression values for each gene. The dendrogram indicates the pattern similarities indicated by Spearman correlation distances (1- Spearman correlation coefficient) and shows a large separation of NCP from NEP and hESC. (C) The expression of early neuronal marker genes was measured in three preparations each of hESC, NEP and NCP by qPCR. The transcript levels of NEP and NCP were calculated relative to hESC. The relative gene expression levels were color coded (significant down-regulation vs. hESC in blue; significant up-regulation in red; non-significant changes marked by “N”. The genes showing different behavior in NEP vs NCP are displayed. All measures of variance and p-values are indicated in the supplemental material.</p

Transcriptional regulation of epigenetic modifiers during neuroepithelial differentiation.

<p>(A) The levels of epigenetic modifier transcripts of NEP and hESC were analyzed by qPCR in three independent cell preparations, and relative abundances were calculated. The data were color-coded, with up-regulated genes displayed in red and down-regulated genes in blue. Measures of variance and p-values are indicated in supplemental material, and only significantly regulated genes are displayed. Genes up-regulated >5-fold are displayed in bold. (B) The transcript levels of HDACs were determined for hESC, NEP, NCP and CTX. All expression levels of differentiated cells were normalized to those of hESC, and relative abundances are displayed. For instance, seven different HDACs were up-regulated in NEP compared to hESC. The dotted lines indicate 2-fold regulation levels. Data are means ± SEM of three independent differentiations. *: p<0.05</p

Effect of neuroectodermal differentiation on localization of histone marks.

<p>(A) hESC were differentiated towards NEP and stained with antibodies specific for Oct4, HNK-1 (neural crest marker), Pax6 (NEP marker) and nestin (neural stem cell marker). Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 100 µm. (B) GO analysis of the up-regulated genes in NEP compared to hESC (C) Whole cell extracts from hESC and NEP were analyzed by Western blot with antibodies specific for the indicated histone H3 modifications. Total histone H3 (Pan-H3) was used as loading control. (D) hESC and differentiated NEP were grown on glass cover slips and immunostained with antibodies specific for H3K9me3 or H4K20me3. The upper panels show grey-scale signal intensities of the stain, the lower panels show a superimposition of the same histone stain as above (red) with a DNA counter-stain (DAPI, blue). Arrows mark two cells with a diffuse H4K20me3 stain, which differs from the spot-like pattern always observed in hESC. Scale bars: 10 µm. (E) Chromatin immunoprecipitiation was perfomed from nuclei of hESC or NEP with antibodies specific for H3K4me3 and H3K27me3. The abundance of promoter regions of <i>OCT4, NANOG, PAX6</i> and <i>SOX2</i> was measured by qPCR with specific primers for the indicated genes. Data were compared to control samples prepared without specific antibody and are indicated as relative enrichment. Data are means ± SD from 2 experiments.</p

Compilation of 150 representative genes involved in epigenetic regulation.

<p>The genes were identified, selected, and classified according to their function based on published literature and database searches. In a further step, the genes related to histone modifications were sub-classified as writers (coding for modifying enzymes such as methyltransferases), as erasers (coding for de-modifying enzymes such as histone deacetylases) and as readers (coding for proteins that bind to the respective modification).</p