Genomic properties of promoter-centered chromatin models
(A) Aggregation plots showing enrichment of genes, SINE and LINE elements around the TSS of genes in different chromatin models. Unique RefSeq TSS were used for analyses. Red curve stands for multi-gene (MG) model, blue for single-gene (SG) model, grey for basal promoter (BP) model, and black dotted line for the rest of the genes (R).
(B) Box-plots showing distribution of percentage GC content of GC isochore around different models, gene length, and intron/exon ratio of RefSeq genes involved in the models. Triple asterisks (***) signifies p-value < 2.2E-16. Red box stands for MG, blue for SG, and grey for BP. Open box is for R (rest of genic regions) as background.
See also Figure S2.

Transcriptional coordination in multi-gene chromatin complexes
(A) Co-localization of multi-gene loci with RNAPII foci. Shown are the nuclear images of RNAPII IF-staining with four randomly-selected multi-gene loci (MG1-4) and 2 control Loci. Representative gene loci are MED20, SYVN1, HIST1, and PLEC1.
(B) Quantitative analysis of nuclei (n=476) and alleles showing overlap of MG loci and RNAPII foci. Percentage overlaps from MG loci and those from control loci are significantly different.
(C) Super multi-gene complex of the histone gene family. Three distant clusters (C1, C2, C3) of HIST1H genes converge together in a super-MG complex. Shown are RNA-Seq, RNAPII and ChIA-PET tracks in MCF7 and K562 cells.
(D) Co-transcription of HIST1H genes in the super-MG complex in (C). Correlation matrix derived from publicly available microarray data of 4,787 samples (Supplemental Information). The rows and columns correspond to genes in each complex and the intervening regions.
(E) RNAPII-bound multi-gene complex at the GREB1 locus. Shown are the ERα-and RNAPII-bound chromatin interactions. Highlighted promoters are anchored by RNAPII, but not by ERα. The bottom panel shows relative interaction frequency by 3C-qPCR data for the perturbation experiments using siERα knockdown and estrogen induction.
(F-G) Time course RT-qPCR following estrogen (E2) induction after siControl (solid) and siERα (dashed) transfections of MCF7 cells. Colors of the curves correspond to genes shown in (E). A secondary axis (red, right side) is used for GREB1 expression to accommodate its high expression level. Expression data of genes involved in the GREB1 multi-gene complex are in (F), and the data for genes outside of the complex are in (G).
See also Figure S4.

Cell-specific chromatin interactions
(A) Contour plots of RNA-Seq data (log RPKM, left) and chromatin interactions (log PET counts, right) in MCF7 and K562 cells, showing common and cell-specific gene expression and chromatin interactions.
(B) Contour plots of interaction data (log PET counts) for genes specifically and commonly expressed in MCF7 and K562 cells.
(C) Enrichment of cell-specific GO terms in genes and chromatin interactions specific in MCF7 and K562 cells. The p-value of 0.01 is marked as dotted line.
(D) An example of K562-specific chromatin interactions. α-globin genes (in dotted line box) interact with distantly located (∼20Kb) DHS sites (highlighted in yellow) which are known to interact with α-globin genes. In sharp contrast, the α-globin genes in MCF7 cells are not expressed and have no interactions with the DHS sites.
(E) An example of MCF7-specific chromatin interactions around the GREB1 locus. The far left highlighted yellow is a RNAPII interaction site that is not overlapped by ERα-bound interactions in this region. It is also the bait site for independent 3C validation of interactions in this region. Tracks included in (D) and (E) are RNA-Seq data, interaction loop view, RNAPII ChIA-PET peaks and interaction PETs, ChIP-Seq density profile of H3K4me1 and H3K4me3, and the ERα-ChIA-PET in (E). The numbers on the right of each track are the highest density value.
See also Figure S6.

Characterization of RNAPII binding peaks and chromatin interactions
(A) RNAPII binding profile around gene body.
(B) Violin plots for intensities of RNAPII peaks involved (red, mean intensity=281) and not involved in interactions (blue, mean intensity=141).
(C) RNAPII-associated chromatin models: basal promoter (BP) with RNAPII binding but no chromatin interaction, single-gene (SG) complex with intra- and/or extra-genic interactions and multi-gene (MG) complex with multiple genes in the interaction clusters. “p” stands for promoter, “g” for gene, and “e” for enhancer, the dotted curve for possible intra-genic loop, and the solid curve for potential loop of enhancer-promoter and promoter-promoter interactions. Data tracks are: 1 and 2, strand specific RNA-Seq data of MCF7 and K562; 3, RNAPII binding peaks and ChIA-PET data. Inset (bottom): DNA-FISH and 3C-qPCR validations of the extra-genic interaction at the KLF4 locus, where the KLF4 promoter and enhancer are ∼1Mb apart. Genomic locations used for 3C bait, test and control sites are indicated. The same locations are also used for DNA-FISH. The numbers (n) of nuclei counted and the fold change (x) in the number of instances showing close proximity (≤ 1μm) are indicated.
(D) Distribution of chromatin models (BP, SG, MG) and the numbers of genes engaged in the models.
See also Figure S1.

Transcriptional activities in RNAPII-associated chromatin models in MCF7 cells
(A) Pie charts of RNAPII binding peaks proximal (blue) or distal (red) to TSS of genes (left), RNA-Seq data for genes with RNAPII peaks near TSS (middle), and RNA-Seq enrichment around inter-genic RNAPII peaks (right).
(B) Correlation of RNAPII binding in basal promoter (BP), single-gene (SG) and multi-gene (MG) models with gene transcription levels measured by RNA-Seq. The RNAPII enrichment heatmap shows binding intensity centered on TSS (±5Kb) along with corresponding gene transcription intensity.
(C) Bar plots of expression levels of genes in the three models (BP, SG, and MG). MG complexes also contain “anchor genes” (TSS proximal to interacting anchors) and “loop genes” (distant from anchors, residing in loop regions). The remaining genes (R) not bound by RNAPII were included as control. Double asterisks (**) indicates significant difference between the mean expressions of genes from SG and MG models (p-value < 4.02E-08).
(D) Expression breadth (number of tissues a gene is expressed in) of genes present in three different chromatin models. P-value is calculated using non-parametric test of Kruskal-Wallis.
(E). Contour plot of log-transformed RNA-Seq RPKM values for co-transcription of interacting genes involved in MG models in MCF7 cells.
(F) Distribution of PCC values for RNAPII- and ERα-bound interacting gene pairs, randomly rewired gene pairs, and randomly picked gene pairs from control regions with the same genomic span and gene density distribution as the multi-gene complex regions.
See also Figure S3.

Epigenomic profiles of chromatin interactions and combinatorial regulation of interacting promoters
(A) Enrichment profiles of TFs and histone modifications centered on RNAPII peaks (±1250bp) of interacting loci of the three models in K562 cells. Solid lines represent “TSS” proximal regions and dotted lines depict “non-TSS” regions. y-axis: sliding median for ChIP-Seq enrichment in the region.
(B) Examples of TF enrichment at RNAPII interacting loci in K562 cells.
(C) Histone modification marks and open chromatin mark (FAIRE) associated with chromatin interaction sites in MCF7 cells. The width of the open boxes in the log ratio track reflects the region where the H3K4me3 and H3K4me1 data were used for the log ratio calculation.
(D) Histograms of normalized H3K4me3/me1 log ratio at RNAPII sites proximal to TSS (TSS) and distal to TSS (non-TSS) of genes in the three chromatin models in MCF7 cells. Two peaks are seen in plot #5 (blue curve for enhancer-like, and the red for promoter-like). The heatmap shows detailed H3K4me3 and H3K4me1 enrichments around RNAPII interaction sites (±5Kb) proximal to TSS. Four distinct clusters, promoter-like (red), enhancer-like (green), heterogeneous (yellow) and weak signals (grey).
(E-G). Reporter gene assay of interacting promoters in MCF7 cells. RNA-Seq, H3K4me3, H3K4me1, H3K4me3/me1 ratio, and RNAPII ChIA-PET data tracks are shown. Numbers on the right side for each track indicate the highest peak intensity.
(E) Promoter-promoter interaction at the INTS1-MAFK locus. The arrow boxes indicate the aligned promoter regions which were cloned in reporter gene constructs for luciferase assay.
(F) Promoter-enhancer-promoter interactions at the C14orf102-CALM1 locus. RNA-Seq data showed that CALM1 was highly expressed, whereas C14orf102 only marginally transcribed (enlarged RNA-Seq track of the C14orf102 locus).
(G) Swap assay of DNA fragments from different multi-gene complexes. The dotted arrow lines show the swap of elements cloned in the distal positions in the reporter gene constructs for luciferase assay.
See also Figure S5.

Long-range enhancers and disease-associated non-coding elements
(A) Percentage difference of enhancer-promoter (EP) and promoter-promoter (PP) interactions in cell-specific vs. common genes from MCF7 and K562 cells. The representation of EP interactions is significantly increased in cell-specific interactions, while the representation of PP interactions is decreased, when compared to interactions that are common to both cell lines.
(B) Proportional distribution of 4 classes of enhancers observed in two cell lines based on locations in relation to gene coding regions. ‘Intra-genic proximal’ enhancers locate inside of gene-body (mostly introns) and interact with the nearby promoters. ‘Extra-genic proximal’ enhancers locate outside of gene body and interact with the nearby promoters. ‘Intra-genic distal’ enhancers locate inside of gene body (mostly introns), bypass nearby genes and interact with faraway gene promoters in long-distance. ‘Extra-genic distal’ enhancers locate outside of gene body, bypass nearby genes and interact with faraway gene promoters in long-distance.
(C) Long-range interactions between SHH (highlighted in yellow, left) and its enhancer located about 1Mb away in an intron of LMBR1 (highlighted yellow, right). The SHH expression is specifically seen in MCF7 cells.
(D) Long-range interactions between IRS1 promoter and two enhancers as well as strong IRS1 expression are seen in MCF7, but not in K562 cells. The dotted line box indicates the enhancer region that contains SNPs associated with insulin resistance, type-2 diabetes (T2D) and coronary artery heart disease identified by a GWAS study. The interactions of enhancer located 1.1Mb away to IRS1 promoter (highlighted in yellow) is validated by DNA-FISH (right). The BAC clones and genomic segments used for DNA-FISH are indicated at the bottom.
Tracks included in (C) and (D) are RNA-Seq density profile, interaction loop view, RNAPII peaks, ChIA-PET interaction PETs, ChIP-Seq density profile of H3K4me1 and H3K4me3 marks.
See also Figure S7.