35 research outputs found
PPARβ is essential for <i>X. laevis</i> development.
<p>(A) Design of the morpholino (PPARβ MO) to target PPARβ translation and of the control morpholino (Co). Capital letters designate nucleotides that can hybridize with the PPARβ MO. (B) Immunoblot showing endogenous PPARβ levels in non-injected embryos (Ni) and embryos injected with PPARβ MO or Co. β-actin served as a loading control. (C) Scoring of A–P axis defects. Different doses of PPARβ MO, Co, or a combination of PPARβ MO and PPARβ_rescue mRNA were injected. Embryos with a length about a third of that of non-injected sibling embryos were scored as ‘very-short axis’, and those with a length of about two thirds of normal were scored as ‘short axis’. (D) Representative not-injected (Ni), Co-injected (Co), MO-injected (MO), and MO combined with rescue injected (PPARβ MO + PPARβ_rescue) embryos.</p
Rate of transcript level variation is maximal at gastrula stage.
<p>(A) Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300-Irie1" target="_blank">[23]</a> were used to quantify transcription variations during normal development. The number of genes showing an RNA level increase or decrease by 2×, 4×, or 8× between two consecutive stages was plotted. Data were normalized by the duration, in hours, of each developmental period analysed. (B) The group of genes with RNA levels that increased 4× or more between stage 11 and stage 13 was considered, and the RNA levels of these genes were plotted at different developmental stages. The rectangles delineate the 25<sup>th</sup> and 75<sup>th</sup> percentiles, the horizontal bar is the median, and the whiskers indicate the 10<sup>th</sup> and 90<sup>th</sup> percentiles.</p
PPARβ interprets a chromatin signature that is deposited at the end of the pluripotent stage.
<p>(A) Seven ‘K27’ genes and eight ‘K4 only’ genes were analysed by ChIP as indicated. Results are presented in a heat map (see also Supplementary Fig. 7). Variation in RNA expression upon PPARβ MO injection was obtained from the RNA-seq data or from qPCR validations. (B) ChIP with H3K27me3 antibody was conducted at stage 9 on 37 ‘PPARβ promoted genes’ and on 27 Control genes. PPARβ promoted genes were chosen among the top 200 most downregulated genes at stage 11, upon MO injection in the list presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300.s008" target="_blank">Table S1</a>, while Control genes did not show a change of expression upon MO injection. Results are presented as percentage of input. The threshold of 1% is indicated. Genes scored as positive for H3K27me3 are indicated by a red dot (see methods for further details on the definition of gene sets and on the criteria of scoring). (C) Sequential ChIPs were conducted. Note that no enrichment was observed for klf11 and for plcg1, which represent negative controls (see panel b). Error is the S.E.M of 2 independent experiments. (D) ChIP using PPARβ antibody was conducted at stage 11.5. Error is the S.E.M of 3 to 4 independent experiments. (E) ChIP with H3K27me3 antibody or PPARβ antibody and <i>q</i>RT-PCR were conducted on embryos treated with DZNep or DMSO and injected with PPARβ MO or Co. Error is the S.E.M of technical replicates of a single experiment that we have replicated with similar results.</p
PPARβ promotes the initiation of differentiation at gastrulation.
<p>(A) Rationale of the transcriptomic analysis of PPARβ loss-of-function. (B) The gene set consisting of predicted direct PPAR target genes in humans <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300-Heinaniemi1" target="_blank">[33]</a> was analysed by GSEA. (C) The Gene Ontology terms or the gene sets that were significantly (FDR<0.2) affected by PPARβ loss-of-function are presented. The gene sets corresponding to germ layer specification are also presented. (D) The gene sets consisting of the 100 most-induced genes and of the 100 most-decreased genes at gastrula (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300.s005" target="_blank">Fig. S5</a>) were analysed by GSEA. FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; NES, Normalized enrichment score.</p
PPARβ promotes differentiation but represses dorsal mesoderm and endoderm specification.
<p>(A)–(C) Embryos were injected with PPARβ MO or Co, allowed to develop until stage 18 (A) or stage 11.5 (B) and (D), and collected for extraction of total RNA. qRT-PCR runs for a selection of neural (blue), mesodermal (red), or endodermal (yellow) markers of differentiation (A) and (B) or of germ layer specification (C) were conducted. RNA levels were normalized to EEF1a and RPL8 and are presented as fold variation between MO and Co samples. Error bars represent the S.E.M. of 3 to 5 independent experiments. (D) Embryos were injected with PPARβ MO or Co, fixed at stg. 11.5, hemi-sectioned along the dorso–ventral axis, and processed for RNA <i>in situ</i> hybridization. While Mo injection did not affect the <i>sox17α</i> expression domain, it resulted in the expansion of <i>brachyury</i> expression dorsally (see the scale) but not ventrally. Arrows indicate the dorsal lip. (E) Quantification of the surface covered by the dorsal and ventral expression domains of <i>brachyury</i> in MO compared to Co hemi-sections. Error bar is the S.E.M. of 10 measurements. *: two-tailed Student’s t-test vs control, P<0.05.</p
Pol II recruitment on SREBP1 target genes is not always synchronized with SREBP1 binding.
<p>(A) The heat-maps represent the recruitment of Pol II to the promoter (left) and to the gene body (middle) of SREBP1 putative target genes along the day, as assessed in our previous ChIP-seq data set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-LeMartelot2" target="_blank">[24]</a>. In parallel, we evaluated hepatic gene expression by microarray analysis in the same samples (right). Hierarchical clustering was done applying a Pearson correlation scores to the data describing Pol II recruitment on the promoters of SREBP1 target genes (left). Three major clusters of genes displaying a different temporal binding profile of Pol II were identified (A1, A2, and A3). The genes are ordered in the three heat-maps according to this clustering. (B) Gene expression data from microarray analysis were fit to a cosine function to estimate the phase of expression (peak time of the fit) of SREBP1 target genes. The graph shows the smoothing of phase distributions of the genes belonging to the three clusters (green line for A1, blue line for A2, magenta line for A3). Only genes with a P-value<0.05 are plotted. Dotted lines define three time intervals containing the most recurrent phases associated to the genes belonging to the clusters A1, A2 and A3. (C) HNF4 binding was tested on randomly selected SRE identified in our SREBP1 ChIP-seq. <i>Gnat1</i> and <i>Anks4b</i> SREs belong to cluster A3 and contain a HNF4 putative binding sites. In contrast, <i>Ldlr</i> and <i>Insig1</i> SREs do not contain in their sequence a HNF4 motif and belong to cluster A2. NEG and POS were used as negative and positive control loci and correspond to two regions of <i>Cyp7a1</i> promoter, localized at −1500 and −150 from the TSS, respectively <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-Kir1" target="_blank">[69]</a>. The graph shows the mean ± SEM of three independent experiments. * indicates P-value<0.01 vs. NEG. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test. Primer sequences are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155.s012" target="_blank">Table S8</a>.</p
Functional annotation clustering of putative SREBP1 targets with a different temporal expression profile.
<p>Enriched GO categories were identified in three distinct sets of rhythmic SREBP1 target genes (P<0.05), based of their phase of expression. To define the three intervals of time we calculated the shortest time range containing the phases of at least 50% of the genes belonging to clusters A1, A2 or A3. For each set, the total number of genes is indicated and the number of genes with annotation is indicated in parentheses. For each functional cluster, only the most significant associated GO term is shown in the table, with the corresponding Modified Fisher Exact P-value.</p
Dynamics of SREBP1 binding.
<p>(A) C57BL/6 mice were fed only during the night (ZT12-ZT24) for one week before collecting liver every 4 hours for one day. Chromatin from 5 mice was pooled at each time point and ChIP with an antibody against SREBP1 was performed. Peaks were positioned where the signal for SREBP1 was at least a four-fold in comparison to the input signal in at least one time point. The heat-map represents SREBP1 binding to all its targets along the time. Hierarchical clustering was done using Pearson correlation scores and identified four major clusters (A, B, C and D). The color scale is indicated below. In the column on the right, black lines indicate that Pol II was detected in the same site as SREBP1 in at least one time point, as assessed in our previous ChIP-seq data set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-LeMartelot2" target="_blank">[24]</a>. (B) Two SREBP1 binding sites were identified in the proximity of <i>Srebp1</i> gene, at a distance of −30 and +299 nucleotides from the <i>Srebp1c</i> and <i>Srebp1a</i> TSS, respectively. Graphs represent the fitting to a cosine function of experimental data obtained on these peaks (black dots), in order to calculate the phase of the binding (dashed line), its interval of confidence (dotted lines) and the associated P-value. (C) Histogram of binding phase frequency in clusters A, B and C, for peaks with a P-value of the amplitude <0.1. None of the peaks belonging to cluster D met this requirement. (D) mRNA expression of <i>Srebp1c</i> was evaluated by qPCR in livers from C57BL/6 mice at the indicated ZT time (n = 5). Data are normalized using 36B4 as housekeeping gene. (E) Hepatic nuclear extracts from C57BL/6 mice were subjected to western blot analysis to detect the nuclear SREBP1. U2AF was used as loading control. Each sample is a pool of 5 livers. (F) Quantification of the Western Blot was performed by densitometry, using ImageJ software.</p
Features of SREBP1 binding sites.
<p>(A) Distribution of peak lengths in the four clusters of SREBP1 binding sites shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen-1004155-g001" target="_blank">figure 1A</a>. (B) Distribution of the distance from the closest annotated TSS of SREBP1 binding peaks belonging to the four clusters. Cluster A is enriched in sequences closer to a TSS, whereas the profile of cluster B, C and D is overlapping with that of 1000 randomly selected sequences (black line). (C) Distribution of the amplitudes of SREBP1 binding oscillation. (D) Overrepresented motifs within SREBP1 binding sites belonging to cluster A were found using MEME <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-Bailey1" target="_blank">[21]</a>. DNA sequences in the window under each SREBP1 peak were used for the sequence analysis. (E) The 236 SREBP1 binding sites (distributed in 223 genes) belonging to cluster A were associated to the closest genes. Annotations are available for 219 of these 224 genes and the Venn diagram shows the overlap between the presence of SREBP1 and the presence of a nearby a binding site for SP1, NFY and/or HNF4. The most relevant functional pathways that were enriched in the different sets of target genes are indicated.</p
Functional annotation clustering of putative SREBP1 targets using DAVID tools.
<p>Enriched GO categories were identified in four distinct sets of SREBP1 target genes exhibiting a different combination of binding sites for SP1, NFY and/or HNF4. In total, 219 out of 223 SREBP1 putative target genes have a functional annotation (the number of annotated genes for each set is indicated in parentheses). The analysis using DAVID groups the GO categories in functional related clusters. For each enriched cellular process, only the most significant associated GO term is shown in the table, with the corresponding Modified Fisher Exact P-value. The complete list of all GO terms enriched in each functional cluster for all the groups is available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155.s008" target="_blank">Table S4</a>.</p