The BAF complex represses basal transcription at the HIV promoter.

<p>(A) Table of subunit composition of the two distinct SWI/SNF complexes, BAF and PBAF, in mammals. (B) GFP expression was monitored by flow cytometry at indicated times after siRNA transfection to measure HIV promoter activity. Results are presented as MFI for cells treated with a control siRNA or siRNAs specific for SWI/SNF subunits. (C) Same experiments as shown in (B) for clone D, with clone E, another Jurkat cell line containing an integrated LTR-GFP virus. Error bars represent the SEM of five independent experiments. * <i>p</i><0.05. (D) Jurkat cells containing an integrated LTR-GFP virus (clone D) were transfected with control siRNA or siRNAs targeting various SWI/SNF complex subunits as indicated. Western blot analysis shows expression of each SWI/SNF subunit after its specific depletion 0, 2, 4, 6, 7, 8, 10, and 14 d after siRNA transfection with each specific antibody and β-actin loading control as indicated.</p

PBAF, recruited by K50K51 acetylated Tat, is a co-factor for Tat activation of the HIV promoter.

<p>(A) J-Lat A2 cells containing an integrated LTR-Tat-FLAG-GFP were stimulated with PMA to induce expression of Tat-FLAG. Tat was immunoprecipitated from untreated or PMA-stimulated cell lysates and its associated proteins were examined by SDS-PAGE and Western blotting with antibodies against the BAF- or PBAF-specific subunits BAF250a and BAF180, and protein kinase D-1 and 14-3-3 as controls. (B) Tat co-immunoprecipitation with BAF180 is modulated by Tat acetylation. Tat (wild-type or K50R/L51R) was immunoprecipitated using anti-FLAG antibody and analyzed by Western blotting using antibody specific for BAF180. Tat acetylation levels were assessed using an anti-acetyl lysine antibody. All proteins were expressed at similar levels under the different experimental conditions as shown by the Inputs. (C) 1G5 Jurkat cells containing integrated LTR-Luciferase (LTR-Luc) were nucleofected with siRNAs against BAF180, BAF250, or with a control siRNA pool. Expression of BAF180, BAF250, and β-actin was analyzed by Western blotting after depletion of either BAF180 or BAF250. (D) Transactivation of the HIV promoter by Tat is reduced in the absence of BAF180. 48 h after siRNA depletion of BAF180 or BAF250, cells were re-transfected with either a control or Tat-expression vector (CMV-driven), and luciferase assay performed after 24 h. Error bars represent the SEM of three independent experiments. * <i>p</i><0.05. (D).</p

Rescue of <i>Tcf3</i> expression in <i>Apc</i>NN ESCs partially restores <i>in vivo</i> neural differentiation.

<p>Teratoma samples were obtained from wild type, <i>Apc</i>NN and <i>Apc</i>NN stably expressing <i>Tcf3</i> (Tcf3 OE) ESCs. Tissue sections were stained by H&E, thionin (marker of cartilage differentiation), and by IHC with specific antibodies against the neural differentiation markers GFAP, 2H3 (neurofilaments) and SV2 (synaptic vesicles). Oct4 IHC analysis was used to asses the presence of undifferentiated EC-like cells in the teratomas.</p

Characterization of Tcf3 over expressing ESCs.

<p>A–B. qRT-PCR (A) and western blot (B) analysis of Tcf3 in <i>Apc</i>NN ESCs stably expressing Tcf3. Wild type and <i>Tcf3</i>^−/− ESCs were used for comparison. Actb was used as an internal control. C. Histogram showing reduction of β-catenin/Tcf reporter activity in <i>Apc</i>NN cells stably expressing Tcf3 (Tcf3 OE) compared to parental <i>Apc</i>NN cells and cells expressing the corresponding empty vector. Luciferase signal from TOP or FOP reporter constructs were measured and TOP/FOP ratios are shown in the graph. Bars represent n = 3 ± SD. D. Histogram showing the percent of alkaline phosphatase (AP) positive colonies formed by plating 500 FACS-sorted cells in N2B27 medium after 7 days. N2B27 medium was supplemented with different combinations of LIF, PD and CHIRON. Two independent <i>Apc</i>NN ESC clones (parental clone and transfected with empty vector) and three independent <i>Apc</i>NN ESC clones expressing <i>Tcf3</i> (Tcf3 OE) were used. Bars represent n = 3 ± SD. E. Histograms showing relative expression of the pluripotency markers <i>Nanog</i> and the early differentiation markers <i>Fgf5</i> in different ESCs cultured for 48 h in N2B27 medium. F. Confocal analysis of ES cells stained with Tuj-1-Alexa 488 and counterstained with the far-red nuclear stain DRAQ5. Wild type, <i>Apc</i>NN and <i>Apc</i>NN expressing <i>Tcf3</i> (Tcf3 OE) ESCs were used in −4/+4 neural differentiation assay and analyzed by immunofluorescence after 13 days of culture. G. Flow cytometric analysis showing expression of the neural progenitor marker Nestin in <i>Apc</i>NN ESCs stably expressing <i>Tcf3</i> (Tcf3 OE) and their control cells (parental <i>Apc</i>NN clone and <i>Apc</i>NN transfected with the corresponding empty vector) or wild type ESCs. Cells were analyzed by the −4/+4 neural differentiation assay and stained with specific antibody against Nestin and Tuj1 after 13 days of culture. Wild type (WT) ESCs are shown as control to indicate the Tuj1 positive population which is absent in other genotypes (0.1% in average in Tcf3 OE clones). Numbers in the graph represent the percent of Nestin-positive cells. For wild type ESCs the Nestin-positive populations before and after excluding the mature neurons are shown. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003424#pgen.1003424.s004" target="_blank">Figure S4</a> for defining different FACS gates.</p

Wnt signaling downregulates Tcf3 expression in mouse ESCs.

<p>A. qRT-PCR analysis of <i>Tcf3</i> in wild type, <i>Apc</i>NN and <i>Apc</i>^Min/Min ESCs. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD. B. Western blot analysis of the core pluripotency markers Oct4, Nanog, Sox2 and Tcf3 on protein lysates isolated from two independent <i>Apc</i>NN clones and wild type control ESCs. Actb and Tubulin were used as an internal control. C–D. qRT-PCR analysis of <i>Tcf3</i> in wild type ESCs treated for different time intervals with Wnt3a conditioned medium (C), or with the GSK-inhibitor SB-216763 (D). L-medium and DMSO were employed as controls, respectively. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD. E. Time course western blot analysis of Tcf3 expression in wild type ESCs treated with Wnt3a conditioned medium (Wnt3a CM) or control L-medium (LM). <i>Actb</i> was used as an internal control. F. qRT-PCR analysis of <i>Tcf3</i> and Wnt target genes Axin2 and Cdx1 in wild type ESC treated for 48 h with different concentrations of GSK-inhibitor SB-216763 or DMSO as control. <i>Actb</i> was used as an internal control; bars represent n = 2 ± SD.</p

Wnt signaling regulates the differentiation potential of mouse ESCs in a dosage-dependent manner.

<p>A. β-catenin/TCF reporter assay in wild type and <i>Apc</i>-mutant ESCs. Measurements are reported as the average luciferase units performed in triplicate for the TOP (filled bars) and FOP (empty bars) reporter constructs (data reported is mean±SD). Numbers in the histogram represent the calculated TOP/FOP ratios. B. Table summarizing the results obtained by teratoma differentiation assay from different <i>Apc</i>-mutant ESCs and their wild type controls. Tissue sections were stained with hematoxylin and eosin (H&E) or used in immunohistochemical analysis using specific antibodies against the neural markers: GFAP, neurofilaments and synaptic vesicles. Adult myosin was used as a mesodermal marker to stain the striated muscle differentiation. Cartilage differentiation was assessed either by H&E or theonin staining. Two independent clones were used for each genotype and differentiation was scored as: (−) not present, (+) weakly present, and (++) present. C. Histogram showing the percent of colonies formed after plating 500 FACS-sorted cells in N2B27 medium supplemented with different combinations of LIF, Mek inhibitor (PD) and GSK-inhibitor (CHIRON). Bars represent mean ± SD, n = 3. D. Dendrogram derived from unsupervised hierarchical clustering of global gene expression in wild type, <i>Apc</i>TT, <i>Apc</i>NT and <i>Apc</i>NN ES cells. Pearson's correlation coefficient and Ward's method were used after MAS 5.0 normalization of all probe sets.</p

The Wnt-regulated miR-211 targets Tcf3 in mouse ESCs.

<p>A. qRT-PCR analysis showing a dosage-dependent up-regulation of miR-211 in different <i>Apc</i>-mutant ESCs. SnoRNA-234 was used as an internal control; bars represent n = 2±SD. B–C. Time course analysis of wild type ESCs treated with Wnt3a conditioned medium (B) or with the GSK-inhibitor SB-216763 (C). L-medium and DMSO were used as controls, respectively. RNAs were isolated at different time points and were subjected to qRT-PCR analysis of miR-211 or snoRNA-234 as an internal control. Bars represent n = 2±SD. D. Western blot analysis of Tcf3 expression in protein lysates isolated from independent clones of wild type ESCs stably expressing miR-211 (miR-211 OE) or the corresponding empty vector (control). Two independent <i>Apc</i>NN clones were included for comparison. E. Schematic representation of the Tcf3-3′-UTR luciferase vector derived from the pmirGLO construct (Promega). Sequence alignment between miR-211 and its target site on Tcf3-3′-UTR. Site directed mutagenesis was used to introduce 7-bp or 4-bp mutations in Tcf3-3′-UTR. F. HEK-293 cells were co-transfected with the Tcf3-3′-UTR luciferase vector, and either with miR-211 or a non-targeting miRNA. Luciferase activity was measured 24 h post-transfection and normalized to Renilla luciferase signal. The same experiment was repeated with the mutant luciferase vectors, MTR1 and MTR2. Asterix represent <i>P</i>-value<0.01 and bars represent n = 3±SEM. G. Flow cytometric analysis of Tuj1 and Nestin in miR-211 over expressing ESCs (miR-211 OE) and their controls (Emp) after 13 days of neural differentiation. Two independent clones were used for each genotype and representative example of each genotype is shown. Numbers in the graph represent the percent of cells in neural (green), progenitor (red) or negative (blue) populations. H. Histogram showing the relative expression of early neural markers <i>Fgf5</i>, <i>Nestin</i>, <i>Pax6</i> and <i>Sfrp2</i> in embryoid bodies derived from independent wild type ESCs clones stably expressing miR-211 or the corresponding empty vector. RNAs were isolated at different time points and were analyzed by qRT-PCR for different lineage markers. Bars represent n = 2±SD. I. qRT-PCR analysis of <i>Fgf5</i>, <i>Nestin</i>, <i>Pax6</i> and <i>Sfrp2</i> in wild type ESCs stably expressing miR-211 or the corresponding empty vector, cultured for 24 h in N2B27 medium. Bars represent n = 2±SD.</p

Regulation of Tcf3 in <i>Apc</i>NN ESCs is associated with histone modifications.

<p>Schematic representation of mouse Tcf3 locus and the different amplicons (P1–P8) analyzed by QPCR in chromatin immunoprecipitation experiment. Chromatin was isolated from <i>Apc</i>NN and wild type ESCs and was immunoprecipitated with specific antibodies against the activating histone marks (H3K4me3 and H3Ac) and the repression histone marks (H3k27me3 and H3K9me3). The input DNA (chromatin before immunoprecipitation) and immunoprecipitated DNA was quantified by QPCR and using specific primers as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003424#s4" target="_blank">materials and methods</a>. Values from each amplicon were normalized to input chromatin and fold change was calculated relative to the corresponding negative region (P1). Bars represent n = 2±SD.</p