<i>NIPBL-AS1</i> does not influence <i>NIPBL</i> transcription.

<p>A) Overview of the genomic position of <i>NIPBL</i> and <i>NIPBL-AS1</i> genes. Strand-specific read coverage of RNA-sequencing data (positive in green; negative in red) from HEK293T cells shows the transcription of <i>NIPBL-AS1</i> antisense to <i>NIPBL</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref001" target="_blank">1</a>]. CTCF binding sites in HEK293 cells (ENCODE hg18) are shown. Primers used in the transcript analysis are indicated as green bars. (B-C) Transcript levels of (B) <i>NIPBL-AS1</i> and (C) <i>NIPBL</i> after antisense oligonucleotide knockdown (ASO2, ASO3) of <i>NIPBL-AS1</i> in HEK293T cells. ASO C was used as control. Transcript levels were normalized against the control sample (ASO C) and the housekeeping <i>SNAPIN</i> using the ΔΔCt method (mean n = 3, error bars +/- s.d., p-values determined with t-Test).</p

Interactions of <i>NIPBL</i> and <i>NIPBL-AS1</i> with a potential distal enhancer.

<p>A) Long-range chromosomal interactions of the <i>NIPBL</i> and <i>NIPBL-AS1</i> promoter detected by chromosome conformation capture (3C-seq) in HEK293T cells using an ApoI digest. The positions of the different viewpoints used are marked in yellow. Three different viewpoints at the promoter (VP4, blue track) and the candidate enhancers regions R1 (VP5, green track) and R2 (R2—VP6, red track) were used. B) CTCF ChIP sequencing track from HEK293 cells (ENCODE). The orientations of the CTCF motifs as determined with JASPAR are shown below the track (red triangle–forward orientation, green triangle–reverse orientation). The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track. C) DNAse clusters as well as histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines (G312878, K562, HeLa-S3, HEMEC, HSMM and HUVEC, available from ENCODE) are displayed as density graph. Black represents areas with the highest enrichment of the signals.</p

Implications for CdLS.

<p>A) Transcript levels of the genes <i>BBX</i>, <i>GLCCI1</i> and <i>ZNF695</i> that were described as dysregulated genes in CdLS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>] and previously confirmed as NIPBL-dependent genes with NIPBL binding sites at the promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>] were analysed in the different enhancer deletion clones D1 and D2 (mean n = 5 for D1 and n = 4 for D2, error bars +/- s.d., p-values determined with t-Test, the transcript levels of the individual clones are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s007" target="_blank">S7 Fig</a>). B) Average transcript levels of <i>NIPBL</i> and <i>NIPBL-AS1</i> in lymphoblastoid cell lines (LCLs) derived from CdLS patients and controls. The details of the four LCL controls and three CdLS LCLs as well as the individual transcript levels are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s008" target="_blank">S8 Fig</a> and in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>]. Two primer pairs for <i>NIPBL</i> and one for <i>NIPBL-AS1</i> were used. Transcript levels were normalized against the housekeeping gene <i>NADH</i> (mean n = 4 for control LCLs and n = 3 for CdLS LCLs, error bars +/- s.d., p-values determined with t-Test).</p