Supplementary document for Evaluation of Slowfade Diamond as a Buffer for STORM Microscopy - 6116375.pdf

Supplementary dat

Comparison of Prep1 Peaks between ES cells and Embryo Trunk.

<p>* RNA-PolII⁺, H3K4Me3⁺ See definitions in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.t001" target="_blank">Table 1</a>.</p><p>^# H3K4Me1⁺, H3K4Me3^-</p><p>^§ from ref [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.ref018" target="_blank">18</a>].</p><p>Comparison of Prep1 Peaks between ES cells and Embryo Trunk.</p

RNA-seq analysis of <i>Prep1</i>^-/- ES cells highlights gene categories not enriched in the total embryo.

<p>The Gene Ontology terms which are enriched in the <i>Prep1</i>^-/- ES cells are shown in (A) whereas the terms enriched only in the fraction of genes bound by Prep1 is shown in (B). The numbers in brackets refer to the number of genes considered. The color code for the <i>p</i>-value is shown. Genes are divided in two groups: down- and up-regulated.</p

ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells

<div><p>The Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was three-fold higher. RNA-seq identifies about 1800 genes down-regulated in <i>Prep1</i>^-/- ES cells which belong to gene ontology categories not enriched in the E11.5 <i>Prep1^i/i</i> differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the <i>Prep1</i>^-/- ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype.</p></div

Growth and differentiation are altered in differentiating <i>Prep1</i>^-/- embryoid bodies (EBs).

<p>(A) Pictures of WT, Prep1-KO1 and Prep1-KO2 embryoid bodies after 2, 5 and 7 days of differentiation. The scale bars indicate 200μm. The EBs were formed starting from 200 cells. (B) RT-qPCR analysis of <i>Oct4</i>, <i>Nanog</i> and <i>Gata4</i> expression in WT, Prep1-KO1 and Prep1-KO2 ES cells (day 0) and in EBs at day 2, day 5, day 7 and day 9 of differentiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#sec009" target="_blank">Methods</a> section). (C) RT-qPCR analysis of <i>Oct4</i>, <i>Nanog</i> and <i>Gata4</i> expression in WT ES cells infected either with a vector containing a scrambled sequence (scr) or with an shRNA targeting <i>Prep1</i>. Measurements were carried out at day 0, day 2, day 5, day 7 and day 9 of differentiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#sec009" target="_blank">Methods</a> section).</p

RNA-seq analysis of <i>Prep1</i>^-/- ES cells highlights gene categories not enriched in the total embryo.

<p>The Gene Ontology terms which are enriched in the <i>Prep1</i>^-/- ES cells are shown in (A) whereas the terms enriched only in the fraction of genes bound by Prep1 is shown in (B). The numbers in brackets refer to the number of genes considered. The color code for the <i>p</i>-value is shown. Genes are divided in two groups: down- and up-regulated.</p

Comparison of Prep1 Peaks between ES cells and Embryo Trunk.

<p>* RNA-PolII⁺, H3K4Me3⁺ See definitions in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.t001" target="_blank">Table 1</a>.</p><p>^# H3K4Me1⁺, H3K4Me3^-</p><p>^§ from ref [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.ref018" target="_blank">18</a>].</p><p>Comparison of Prep1 Peaks between ES cells and Embryo Trunk.</p

Prep1 reconstitution rescues growth and differentiation of <i>Prep1</i>^-/- Embryoid bodies (EBs).

<p>(A) Prep1 KO1 ES cells where infected either with an empty vector (EV) or with a vector containing Prep1 cDNA. Their ability to form EBs was assessed along with WT ES cells infected with an empty vector. Pictures of WT-EV, Prep1 KO1-EV and Prep1 KO1-Prep1 EBs after 2, 5, 7 and 9 days of differentiation. The scale bars indicate 200μm. The EBs were formed starting from 1000 cells. The histogram to the right shows the number of EBs formed at different differentiation times in the different cell lines. (B) RT-qPCR analysis of <i>Oct4</i>, <i>Gata4</i> and <i>Fgf4</i> RNA in WT-EV, Prep1 KO1-EV and Prep1 KO1-Prep1 ES cells (day 0) and differentiating EBs at day 2, day 5, day 7 and day 9 of differentiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#sec009" target="_blank">Methods</a> section). (C) RT-qPCR analysis of <i>Fgfr2</i> and <i>Fgfr3</i> RNA in WT-EV, Prep1 KO1-EV and Prep1 KO1-Prep1 ES cells (day 0) and differentiating EBs at day 2, day 5, day 7 and day 9 of differentiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#sec009" target="_blank">Methods</a> section).</p

ChIP-seq analysis of Prep1 binding sites in ES cells and comparison to the E11.5 embryo trunk.

<p>(A) Subdivision of the Prep1 peaks among peaks -500 to + 100 bp to TSS (TSSA, Transcription Start Site-Associated) with or without promoter marks (H3K4Me3⁺, RNA-Pol-II⁺) (PROM), IG (intragenic), CI (Close-intergenic; less than 20 Kb from a TSS), FI (Far Intergenic; more than 20 Kb from a TSS). We only considered ChIP-seq peaks with p-value <10^-6 (based on the comparison between immunoprecipitated and non immunoprecipitated input samples). The information on the presence of RNA polymerase II and of the indicated histone modification was taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.ref021" target="_blank">21</a>]. (B) Core Prep1 binding motif (DECA) identified in 70% of all (3902) ES cells peaks. (C) Venn diagrams of ChIP-seq peaks for ES cells and total embryo (left panel) and of the genes corresponding to these peaks (right panel). (D) Promoter and enhancer peaks distribution in ES total, ES^exc, Embryo total and Embryo^exc peaks. Definition of promoters and enhancers as described in the text. The data for the embryo (E11.5 embryo trunks) are taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.ref018" target="_blank">18</a>]. (E) Boxplot and whisker diagrams of the scores of the ES cell-exclusive peaks (ES^exc) and ES cell-embryo common peaks (ES-embryo com) grouped in TSSA (Transcription Start Site-Associated), IG (Intragenic), CI (Close-Intergenic) and FI (Far Intergenic) regions.</p

<i>Prep1</i>^-/- Embryoid bodies (EBs) express altered levels of various <i>Wnt</i> RNAs during differentiation and Prep1 directly targets the <i>Wnt3</i> promoter.

<p>(A) Deficient regulation of Wnt genes during differentiation of <i>Prep1</i>^-/- EB. RT-qPCR analysis of <i>Wnt</i> mRNA expression. Fold change variations of <i>Wnt3</i>, <i>Wnt3a</i>, <i>Wnt4</i>, <i>Wnt6</i>, <i>Wnt7a</i>, <i>Wnt7b</i>, <i>Wnt8a</i>, <i>Wnt8b</i> and <i>Wnt9b</i> RNAs in Prep1 WT v. KO1 ES cells at day 0, day 2 and day 5 of differentiation. (B) Chromatin Immunoprecipitation (ChIP) analysis of the Wnt3 promoter. Region 1 contains the Prep1 binding site, while Region 2 does not (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#pone.0122518.s003" target="_blank">S3 Fig</a> for details on the sequence). The fold enrichment over input obtained by immunoprecipitation with an anti-Prep1 antibody in WT ES cells is shown for region 1 and region 2. As negative controls we used either IgG with WT ES cells chromatin, or anti-Prep1 antibody in the <i>Prep1</i>^-/- KO1 ES cells chromatin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122518#sec009" target="_blank">Methods</a> section). (C) Immunobloting of total cell lysates from the WT ES cells (WT), <i>Prep1</i>^-/- ES cells transfected with empty vector (KO-EV), Prep1-rescued ES cells (KO-Prep1) before (D0) and after 5 days of differentiation of EB (D5) using an anti-active beta-catenin antibody (anti-ABC). Anti-vinculin antibody was used as loading control. The quantification of the immunobloting is shown in the right panel. Relative units (R.U.) are the band intensities normalized to that of the band of ES WT cells before differentiation.</p