A Cohesin-Independent Role for NIPBL at Promoters Provides Insights in CdLS
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 [31]. 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 [33]. 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.