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

Binding of NIPBL, cohesin and CTCF in the human genome.

<p><b>A</b> Genomic binding of NIPBL, CTCF and the cohesin subunits SMC3 and SMC1A in the breast endothelial cell line HB2 at a selected region of chromosome 19 as determined by ChIP-sequencing. The RNA Pol II binding profile, the control ChIP and the RNA-sequencing data from these cells are also shown. <b>B–D</b> Heatmaps showing the ChIP signal intensity of the indicated ChIP-sequencing experiments in a window of +/−500 bp around all NIPBL peaks (<b>B</b>) as well as the top 10000 CTCF (<b>C</b>) and SMC3 (<b>D</b>) peaks. Cohesin (SMC3, SMC1A) and CTCF binding does not correlate with NIPBL binding events. RNA Pol II signals are found near NIPBL, consistent with the localization of NIPBL at promoters. Cohesin binding events correlate well between SMC3 and SMC1a and with CTCF. Peaks are ranked by size with the strongest peaks at the bottom of the graph. <b>E</b> ChIP was performed with NIPBL#1, SMC3 and control antibodies from HB2 cells and analyzed by qPCR with primers specific for cohesin, NIPBL and a negative (<i>AMY</i>) sites. NIPBL ChIP signals on cohesin sites are at background level (red horizontal line). Only the DUSP10 site is higher than the background in the SMC3 ChIP, very likely due to a CTCF/cohesin site close to the NIPBL site. All experiments were at least performed three times and one representative example is shown.</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

<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