List of upregulated genes on 12p in PKS probands.

<p>List of upregulated genes on 12p in PKS probands.</p

Ingenuity pathway analysis of dysregulated genes in PKS.

<p>Green circles represent the genes down-regulated in PKS probands, and red circles represent the genes up-regulated in PKS probands.</p

BOX plot of HOX gene clusters.

<p>HOX A is down- and HOX B Cluster is up-regulated in PKS patients.</p

Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells with Distinct Mutations in <i>SMC1A</i> and <i>SMC3</i> Genes

Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS, we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, <i>SMC1A</i> and <i>SMC3</i>. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the <i>c-MYC</i> gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS

Schematic illustration of disease mechanism of PKS.

<p>Schematic illustration of disease mechanism of PKS.</p

Patient and control sample clustering.

<p>Red squares/circles represent PKS patients and Green squares/circles represent control samples. (A) Unsupervised clustering of 26 samples using all genes. (B) Unsupervised clustering of 26 samples using genes located on 12p.(C) PCA result. Proportion of Variance % (PC1-24.818, p 0.001; PC2-17.814, p 0.022; PC3-0.022, p 0.772).</p

Sample/mosaic ratio information.

<p>Sample/mosaic ratio information.</p

Gene expression levels in PKS.

<p><b>a</b>: Mosaic 12p expression level correlation: Average expression of 171 genes on 12p in patients and controls. Red bars are patients and Grey is controls. X-axis represents samples and Y-axis shows average expression of genes on 12p. <b>b</b>: Level of i12p mosaicism compared to 12p gene expression: average of 64 genes with expression higher than the 3rd quartile of global expression distribution, were correlated to mosaicism%. The blue line in the figure is the fitting line of linear regression. Red diamonds are the probands and green are the controls.</p

NIPBL binds to active promoters together with other transcription factors.

<p><b>A</b> Binding of NIPBL, CTCF and cohesin (SMC3) relative to active genes in HB2 cells. The different regions were defined as follows; upstream: −5 kbp to −1 kbp from transcription start sites; promoter: 1 kbp upstream and downstream from TSS; gene body: +1 kbp from TSS until end of the coding sequence; downstream: end of the coding sequence - +5 kbp (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004153#pgen.1004153.s008" target="_blank">Table S2</a>). <b>B</b> Bubble plot representation of NIPBL binding around RNA Pol II peaks in HB2 cells. The x-axis denotes the position of NIPBL respective to the closest RNA Pol II peak and the y-axis the strength of the RNA Pol II peak. Bubble size indicates the strength of the NIPBL peak. NIPBL binds 100–250 bp around RNA Pol II peaks, preferentially upstream, which is consistent with binding to active promoters. <b>C</b> NIPBL binding in the control LCL's (N5) was compared with localization of histone modifications and CTCF in the lymphoblastoid cell line GM12878 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004153#pgen.1004153-Ernst1" target="_blank">[31]</a>. The plot is centred on the NIPBL peaks and the y-axis displays the signal intensity of the respective histone modification and CTCF in GM12878 cells. <b>D</b> Heatmap correlating the P300 ChIP signals +/−500 bp around P300 binding sites observed in GM12878 cells with the sequencing reads obtained for the control and for NIPBL ChIP in control (N5) and patient cells (PT9). The plot is centred on the 10000 strongest P300 peaks clustered into different genomic regions as in (A). <b>E</b> Identical heatmaps generated for the RAD21 peaks observed in GM12878 cells. <b>F</b> Consensus motif derived de-novo from NIPBL binding sites in HB2 cells. The region ±50 bp around the peak maximum was used to determine motifs with MEME <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004153#pgen.1004153-Bailey1" target="_blank">[33]</a>. These motifs are nearly identical to the respective motifs of the transcription factors NFYA and SP1, indicating that one or more transcription factors might colocalize with NIPBL. <b>G</b> Binding of NFYB to NIPBL sites as discovered by the motif analysis in (D) and the comparison to binding sites of other transcription factors in (E) was confirmed by ChIP-qPCR with anti-NFYB antibodies. <b>H</b> Heatmaps comparing +/−500 bp around NIPBL sites observed in LCL's (N5) with ChIP-sequencing data of various transcription factors (GM12878 cells) revealed a subset of transcription factors colocalizing with NIPBL. The heatmaps reveal a strong correlation of PBX3, SP1, C-FOS, IRF3 and NFYA/B with NIPBL sites. <b>I</b> Heat maps showing the correlation of the factors in (H) to NFYB sites at GpG island promoters (sites at CpG island promoters ranked according to strength with the strongest signals at the bottom). The strongest correlation with the other factors is visible for the strongest NFYB peaks.</p

NIPBL is important to maintain gene activity.

<p>Transcript levels of genes with NIPBL-bound promoters and no cohesin sites close to the gene (<i>GLCCI1</i>, <i>BBX</i>, <i>TSPAN31</i>, <i>ARTS-1</i> and <i>ZNF695</i>) and the cohesin-regulated <i>MYC</i> gene were analyzed by RT-PCR/qPCR after RNAi depletion of NIPBL, MAU2 or SMC3 in HB2 cells. The cells were synchronized in G2 phase and the transcript levels are normalized against the housekeeping gene <i>NAD</i>. Transcripts of NIPBL, MAU2 and SMC3 were also analyzed to exclude that NIPBL affects transcription of MAU2 and SMC3 and vice versa. All three genes serve also as negative control genes without NIPBL binding site at the promoter, although MAU2 and SMC3 have intronic cohesin binding sites. P-values were determined using Students test using between 3 and 9 independent biological replicates. The p-value and number of replicates is indicated for each graph. Values that are significantly different (P-value<0.05) from control RNAi are highlighted in red. (error bars ± s.d.).</p