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

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

Schematic illustration of disease mechanism of PKS.

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

Chromatin association of NIPBL, cohesin and CTCF during exit from mitosis.

<p><b>A</b> To address the association of cohesin, CTCF and NIPBL with chromatin during end of mitosis HeLa cells were fixed with PFA and stained with antibodies against CTCF (CTCF#1), the cohesin subunit RAD21 and NIPBL (NIPBL#2). Image stacks were taken with a confocal microscope and a Z-projection generated with Image J. Cells in interphase and different stages of mitosis are shown, from top to bottom: interphase, metaphase, late anaphase, telophase, completed cytokinesis together with a metaphase. <b>B</b> One image slice (100 µm) of the telophase images in (<b>A</b>) is shown to highlight the lack of cohesin signal on chromatin while NIPBL and CTCF are already present.</p