An oestrogen-receptor-alpha-bound human chromatin interactome

Abstract

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

Figure 1. ChIA-PET method with validations

(a) ChIA-PET schematic. DNA fragments in sonicated, ChIP-enriched chromatin complexes were processed by linker ligation, proximity ligation, PET extraction, sequencing, and mapping to reveal interacting loci. (b) ChIA-PET browser tracks: 1, H3K4me3 ChIP-Seq; 2, RNAPII ChIP-Seq; 3, ERα (orange) and FoxA1 ChIP-chip (green); 4, ERα ChIA-PET density ; 5, Inter-ligation PETs. Inset: 3C validation of interacting ERαBS (purple) and controls (blue) under ethanol (ET) and oestrogen-induction (E2). (c) 4C validation, showing 4C bait region (blue) and interaction targets (purple bars). (d) FISH validation, showing increased P2/P1 interactions under E2-induction with background normalization (P3/P2). FISH probe genomic locations (P1/P2/P3) are indicated.

Figure 2. ERαBS reproducibility and association with chromatin interactions

(a) Numbers of ERαBS identified with different ChIP enrichment cutoffs and reproducibility analyses as measured by overlapping with another ChIA-PET dataset (IHH015F), ChIP-Seq, and ChIP-chip data. Examples of ERαBS involved in (b) complex-interactions, (c) duplex-interactions, and (d) no-interactions (singleton inter-ligation PETs only or no inter-ligation PETs). (e) ERαBS distribution in different categories of interactions as exemplified in b-d.

Figure 3. Association of ERα-bound chromatin interactions with functional marks

(a) Association of ERαBS in complex-, duplex-, and no-interaction categories with RNAPII (red), H3K4me3 (blue), and FoxA1 (green) functional marks. (b) Association of proximal and distal interacting and non-interacting ERαBS with H3K4me3 and RNAPII functional marks.

Figure 4. Proposed ERα-bound chromatin interaction and transcription regulation mechanism

(a) Distal ERαBS interact with proximal sites, forming chromatin loops. Anchor genes (green and blue) are close to interaction anchors with concentrated active transcriptional machinery (red shading). Other genes far from interaction centers (grey) are less active. (b) Expression microarray data (oestrogen induction from 0 to 48h; red denotes activation; green denotes repression) for interaction anchor genes, loop genes, and genes near no-interaction ERαBS, with all other UCSC Genes (“All genes”) denoting background. (c) ChIA-PET interactions data at the FOS/JDP2/BATF loci. Transcription activities are shown by H3K4me3/RNAPII ChIP-Seq and RT-qPCR analysis (oestrogen induction from 0 to 24h).

Figure 5. ERα-bound chromatin interactions are required for transcription activation

Genome browser at the GREB1 locus showing data tracks: H3K4me3 and RNAPII ChIP-Seq (1 and 2); RNAPII ChIP-qPCR scans (3 and 4) using different RNAPII antibodies under oestrogen-induction (E2, in red) and ethanol control (ET, in grey); ERα (orange) and FoxA1 (green) ChIP-chip (5); ChIA-PET density (6) and interaction data (7). Inset: siRNA knockdown experiments. MCF-7 cells were transfected with siRNA against ERα (siERα) or control (siCtrl), and then analyzed by (a) western blot with ERα and calnexin (control) antibodies; (b) RT-qPCR on GREB1 expression; and (c) 3C assays at GREB1: siERα knockdown abolishes chromatin interactions and turns off transcription.