Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 13;10(2):e1004153.
doi: 10.1371/journal.pgen.1004153. eCollection 2014 Feb.

A cohesin-independent role for NIPBL at promoters provides insights in CdLS

Jessica Zuin  1 , Vedran Franke  2 , Wilfred F J van Ijcken  3 , Antoine van der Sloot  3 , Ian D Krantz  4 , Michael I J A van der Reijden  1 , Ryuichiro Nakato  5 , Boris Lenhard  6 , Kerstin S Wendt  1
Affiliations

A cohesin-independent role for NIPBL at promoters provides insights in CdLS

Jessica Zuin et al. PLoS Genet. .

Abstract

The cohesin complex is crucial for chromosome segregation during mitosis and has recently also been implicated in transcriptional regulation and chromatin architecture. The NIPBL protein is required for the loading of cohesin onto chromatin, but how and where cohesin is loaded in vertebrate cells is unclear. Heterozygous mutations of NIPBL were found in 50% of the cases of Cornelia de Lange Syndrome (CdLS), a human developmental syndrome with a complex phenotype. However, no defects in the mitotic function of cohesin have been observed so far and the links between NIPBL mutations and the observed developmental defects are unclear. We show that NIPBL binds to chromatin in somatic cells with a different timing than cohesin. Further, we observe that high-affinity NIPBL binding sites localize to different regions than cohesin and almost exclusively to the promoters of active genes. NIPBL or cohesin knockdown reduce transcription of these genes differently, suggesting a cohesin-independent role of NIPBL for transcription. Motif analysis and comparison to published data show that NIPBL co-localizes with a specific set of other transcription factors. In cells derived from CdLS patients NIPBL binding levels are reduced and several of the NIPBL-bound genes have previously been observed to be mis-expressed in CdLS. In summary, our observations indicate that NIPBL mutations might cause developmental defects in different ways. First, defects of NIPBL might lead to cohesin-loading defects and thereby alter gene expression and second, NIPBL deficiency might affect genes directly via its role at the respective promoters.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chromatin association of NIPBL, cohesin and CTCF during exit from mitosis.
A 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 One image slice (100 µm) of the telophase images in (A) is shown to highlight the lack of cohesin signal on chromatin while NIPBL and CTCF are already present.
Figure 2
Figure 2. Binding of NIPBL, cohesin and CTCF in the human genome.
A 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–D Heatmaps showing the ChIP signal intensity of the indicated ChIP-sequencing experiments in a window of +/−500 bp around all NIPBL peaks (B) as well as the top 10000 CTCF (C) and SMC3 (D) 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. E 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 (AMY) 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.
Figure 3
Figure 3. NIPBL binds to active promoters together with other transcription factors.
A 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 Table S2). 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. C NIPBL binding in the control LCL's (N5) was compared with localization of histone modifications and CTCF in the lymphoblastoid cell line GM12878 . 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. D 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). E Identical heatmaps generated for the RAD21 peaks observed in GM12878 cells. F 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 . 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. G 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. H 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. I 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.
Figure 4
Figure 4. NIPBL is important to maintain gene activity.
Transcript levels of genes with NIPBL-bound promoters and no cohesin sites close to the gene (GLCCI1, BBX, TSPAN31, ARTS-1 and ZNF695) and the cohesin-regulated MYC 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 NAD. 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.).
Figure 5
Figure 5. Position of NIPBL sites is conserved but the occupancy is reduced in CdLS.
A NIPBL ChIP-sequencing data of a region of chromosome 19 showing that NIPBL sites are conserved between CdLS patient cells and the control (M – Megabases). B CdLS patient and control cell lines used in this study. The cell lines highlighted were used for ChIP-sequencing. The tables were derived from . Nucleotide numbering refers to the NIPBL B isoform cDNA sequence with GeneBank accession number NM_015384 and starting at the +1 position of the translation initiation codon. C Venn diagrams indicating the number of NIPBL binding sites observed in the different LCL's and also the sites consistently called in all three lines. The majority of binding sites is conserved, although each cell line displays cell-line specific sites. D NIPBL binding is reduced in LCL's derived from CdLS patients. NIPBL ChIP was performed for four patient-derived cell lines and four age and gender-matched controls and qPCR analysis was performed for seven NIPBL binding sites and one cohesin site. The enrichment compared to control IgG ChIP was calculated. The data for the individual cell lines are displayed in Suppl. Fig. S6. Here we present the average relative enrichment for all control and patient-derived lines, p-values derived with a Student test are indicated above the respective columns.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Peters JM, Tedeschi A, Schmitz J (2008) The cohesin complex and its roles in chromosome biology. Genes Dev 22: 3089–3114. - PubMed
    1. Sjogren C, Strom L (2010) S-phase and DNA damage activated establishment of sister chromatid cohesion–importance for DNA repair. Exp Cell Res 316: 1445–1453. - PubMed
    1. Wendt KS, Yoshida K, Itoh T, Bando M, Koch B, et al. (2008) Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451: 796–801. - PubMed
    1. Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, et al. (2008) Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132: 422–433. - PubMed
    1. Stedman W, Kang H, Lin S, Kissil JL, Bartolomei MS, et al. (2008) Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators. Embo J 27: 654–666. - PMC - PubMed

Publication types

MeSH terms

-