ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters

Abstract

Chromatin interactions connect distal regulatory elements to target gene promoters guiding stimulus- and lineage-specific transcription. Few factors securing chromatin interactions have so far been identified. Here, by integrating chromatin interaction maps with the large collection of transcription factor-binding profiles provided by the ENCODE project, we demonstrate that the zinc-finger protein ZNF143 preferentially occupies anchors of chromatin interactions connecting promoters with distal regulatory elements. It binds directly to promoters and associates with lineage-specific chromatin interactions and gene expression. Silencing ZNF143 or modulating its DNA-binding affinity using single-nucleotide polymorphisms (SNPs) as a surrogate of site-directed mutagenesis reveals the sequence dependency of chromatin interactions at gene promoters. We also find that chromatin interactions alone do not regulate gene expression. Together, our results identify ZNF143 as a novel chromatin-looping factor that contributes to the architectural foundation of the genome by providing sequence specificity at promoters connected with distal regulatory elements.

Figure 1. ZNF143 binds promoters and occupies CTCF and cohesin bound distal regulatory elements.

(a) A heatmap of the signal intensities from ChIP-seq assays against ZNF143, CTCF, SMC3 and POL2 across all ZNF143-binding sites (± 5 kilobases (kb)) called in GM12878 cells. (b) Violin plots of the signal intensities from ChIP-seq assays against ZNF143, CTCF, SMC3 and POL2 at their respective binding sites and the distributions of these sites across chromatin states defined by epigenetic modifications. The violin plots are split to show the distribution of the top decile of each factor separately. Enh, Enhancer; Ins, Insulator; Pro, Promoter; Tx, Transcription. (c) A bar plot revealing the fraction of ZNF143 chromatin-binding sites in GM12878 cells that harbour its DNA recognition sequence. (d) The average binding intensity of ZNF143, CTCF, SMC3, and POL2 at POL2-bound promoters (top) and CTCF-binding sites (bottom).

Figure 2. ZNF143 preferentially binds at chromatin interaction anchors.

(a) ZNF143-binding sites across the genome are enriched within the anchors of chromatin interactions reported in 5C assays. The normalized enrichment of ZNF143 and other transcription factors at both ends (anchors) of chromatin loops identified by 5C assays in GM12878 cells is shown. Box plots represent the normalized null distribution derived from the comparison between chromatin interactions and 1,000 RMBSs. Red dots indicate the observed per cent overlap of the transcription factor-binding sites within both 5C interaction anchors value relative to the generated null distribution represented as z scores. (b) Venn diagram depicting shared versus cell type-specific ZNF143-binding sites identified by ChIP-seq assays in GM12878, K562 and HelaS3 cells. (c) The above panel shows the percentage of cell type-specific chromatin interactions defined by 5C assays that harbour a DNaseI hypersensitivity site (DHS) bound by ZNF143 specifically in GM12878, K562 or HelaS3 cells. The bottom panel represents the proportion of promoters (± 2.5 kilobases (kb) from the transcription start site) of genes uniquely expressed in GM12878, K562 or HelaS3 bound by ZNF143 specific to one of these cell lines (G: GM12878, K: K562, H: HelaS3). The P value is derived from a χ²-test; (NS) not significant; *P≤0.05; ***P≤0.001. (d) Signal intensities for 10 different epigenetic modifications profiled by ChIP-seq in GM12878 (red), K562 (blue) and HelaS3 (green) cells across the unique top decile ZNF143-binding sites reported in GM12878 (top panel), K562 (middle panel) and HelaS3 (bottom panel) cells. The shaded area represents the s.e.m.

Figure 3. ZNF143 is required for the formation of chromatin interactions.

(a) Chromatin interactions predicted by the IFC analysis anchored on the TBL1XR1 gene promoter are represented by Bezier curves. Signal and peak files for ZNF143, SMC3, RAD21 and CTCF defined by ChIP-seq assays in HelaS3 are presented. Test (t1 and t2) regions (black boxes) and negative control (nc1–5) regions (grey boxes) are shown. (b) 3C assays anchored at the TBL1XR1 gene promoter reveal the interactions frequencies at a number of predicted chromatin interactions in HelaS3 cells transfected with the siRNA control (green bars). These interactions are diminished on silencing ZNF143 (grey bars). (c) ChIP-qPCR assays against ZNF143 at the TBL1XR1 gene promoter (proximal) and distal site (t1) mapping to the chromatin interactions are presented in HelaS3 cells transfected with the siRNA control (green bars). The ChIP signal is diminished on silencing ZNF143 (grey bars). (d) RT–qPCR assays reveal the expression of the TBL1XR1 gene in HelaS3 cells transfected with the siRNA control (green bars) and on silencing ZNF143 (grey bars). (e–h) Similar to a–d but for the EEF1A1 gene locus The P value is derived from a t-test; *P≤0.05; **P≤0.01. t1=test region (black boxes); nc1–4=negative control regions (grey boxes). Error bars indicate the s.e.m. Experiments were performed in triplicate. rel., relative.

Figure 4. Genetic variants modulate ZNF143 binding to the chromatin changing the frequency of chromatin interactions.

(a) Position of the rs2232015 SNP with regards to one of the ZNF143 DNA recognition sequences (motif 1). (b) Position of the rs13228237 with regards to the second ZNF143 DNA recognition sequence (motif 2). (c) Location of the rs2232015 SNP with respect to the binding profiles of ZNF143, SMC3, RAD21 and CTCF in GM12878 cells (left panel). Allele-specific bias in the ZNF143 ChIP-seq reads at the rs2232015 SNP is shown (right panel). The number of reads mapping to the positive strand (solid grey) and negative strand (dashed grey) are also shown for both the reference and variant allele. (d) Same as for c but for the rs13228237 SNP. (e) Allele-specific ChIP-qPCR against ZNF143 and 3C-qPCR results at the rs2232015 SNP in GM12878 cells are presented. Bar charts illustrate the bias in the allele ratio for the rs2232015 SNP in both assays. Error bars indicate the s.e.m. Experiments were performed in triplicate. Results from the eQTL analysis in lymphoblastoid cells are presented for the PRMT6 gene. Expression values are plotted by genotype. Expression values are presented as probabilistic estimation of gene expression residuals (PEER) normalized reads per kilobase of transcript per million mapped reads (RPKM). (f) Same as in e but relevant to the rs13228237 SNP. Error bars indicate the s.e.m.

Figure 5. Schematic representation of chromatin interactions involving gene promoters.

ZNF143 contributes the formation of chromatin interactions by directly binding the promoter of genes establishing looping with distal element bound by CTCF.