RUNX1 contributes to higher-order chromatin organization and gene regulation in breast cancer cells

Abstract

RUNX1 is a transcription factor functioning both as an oncogene and a tumor suppressor in breast cancer. RUNX1 alters chromatin structure in cooperation with chromatin modifier and remodeling enzymes. In this study, we examined the relationship between RUNX1-mediated transcription and genome organization. We characterized genome-wide RUNX1 localization and performed RNA-seq and Hi-C in RUNX1-depleted and control MCF-7 breast cancer cells. RNA-seq analysis showed that RUNX1 depletion led to up-regulation of genes associated with chromatin structure and down-regulation of genes related to extracellular matrix biology, as well as NEAT1 and MALAT1 lncRNAs. Our ChIP-Seq analysis supports a prominent role for RUNX1 in transcriptional activation. About 30% of all RUNX1 binding sites were intergenic, indicating diverse roles in promoter and enhancer regulation and suggesting additional functions for RUNX1. Hi-C analysis of RUNX1-depleted cells demonstrated that overall three-dimensional genome organization is largely intact, but indicated enhanced association of RUNX1 near Topologically Associating Domain (TAD) boundaries and alterations in long-range interactions. These results suggest an architectural role for RUNX1 in fine-tuning local interactions rather than in global organization. Our results provide novel insight into RUNX1-mediated perturbations of higher-order genome organization that are functionally linked with RUNX1-dependent compromised gene expression in breast cancer cells.

Keywords: Breast cancer; Hi-C; MCF-7; RUNX1; TAD; Topologically associating domain.

Figure 1

RUNX1 knockdown results in aberrant gene regulation in MCF-7 cells. (A) Western blot of the RUNX1 protein levels of shNS and shRNX1 MCF-7 cells. There is a near complete knock down of RUNX1 protein levels. (B) Scatter plot showing the expression values for shNS and shRUNX1 cells. The red and blue dots denote the significantly up- and down-regulated genes, respectively. (C–D) Bar graph showing the −log10 p-values for the top 5 REACTOME terms for the genes that are (C) down- and (D) up-regulated upon RUNX1 knockdown.

Figure 2

RUNX1 ChIP-seq in parental MCF-7 cells. (A) Pie chart showing the distribution of RUNX1 ChIP-seq peak annotation. (B) Normalized RUNX1 ChIP-seq signal intensity plot for all human UCSC genes +/− 2kb. (C) MEME de novo motif analysis of the RUNX1 peaks. The peaks are ordered by significance from top to bottom. (D–E) Two examples of ChIP-seq genome browser views of RUNX1 binding and the input control, along with shNS and shRUNX1 RNA-seq tracks for (D) MALAT1 and (E) NEAT1 lncRNA genes. (F) Bar graph showing RUNX1 peak binding +/− 5kb of transcriptional start site (TSS), or +/− 20kb of the gene bodies of up- and down-regulated genes.

Figure 3

RUNX1 is enriched at TAD boundaries. (A) Venn diagram showing that the TAD boundaries are largely similar between shNS and shRUNX1 Hi-C datasets. (B) Pie chart showing the percentage of RUNX1 localization at TAD boundaries (left panel) and the Venn diagram showing the overlap of RUNX1 overlapping TAD boundaries (right panel). 84% of all RUNX1 bound TAD borders are overlapping between shNS and shRUNX1 cells. If a RUNX1 peak overlapped with a TAD boundary (>1bp), it was considered as being located at that TAD boundary. (C) The frequency plot of RUNX1 ChIP-seq peaks per 25kb for +/− 1Mb of every TAD boundary. (D) An example region on chr10 showing three different TADs, along with their chromatin states assessed by H3K27ac and H3K27me3 ENCODE ChIP-seq tracks (from University of Washington), shNS TAD borders and RUNX1 ChIP-seq peak binding at the TAD boundary regions.

Figure 4

RUNX1 knockdown results in the perturbation and the emergence of many long-range interactions. The significant differential interactions at 1Mb resolution are shown for each chromosome versus itself. The z-score differences of shRUNX1/shNS Hi-C matrices for each chromosome denote the interactions that are either increased (red color), or decreased (blue color).

Figure 5

RUNX1 knockdown results in extensive loss and gain of long-range interactions at specific loci. (A) Interaction heatmap of the BMP2 gene locus (indicated by an arrow) +/− 5Mb on chr20. The top and the bottom parts of the heatmap show the interaction frequencies in the shRUNX1 and shNS cells, respectively. The color scale represents the number of normalized reads. (B) Heatmap of the same region showing the differential interactions between shRUNX1 and shNS. (C) Heatmap showing the significantly altered interactions around the BMP2 gene locus. In the lower panel, the UCSC genes, the MCF-7 ChIP-seq signal for RUNX1 from this study, ChIP-seq for CTCF, RAD21 as well as DHS from the ENCODE database are shown. The highlighted region indicates the BMP2 gene.