Background: Transcription factors (TFs) driving the divergence between ""proliferative"" and ""stem-like"" hybrid epithelial/mesenchymal (E/M) states in triple-negative breast cancer (TNBC) remain poorly understood.
Methods: PPARD was identified as a top-ranked candidate driver through a CRISPRa screening in MDA-MB-231 cells. Screening data were processed using the MAGeCK-RRA. For ChIP-seq data, paired-end reads were aligned via Bowtie2, and reproducibility was quantified using the Irreproducible Discovery Rate (IDR) framework. Only high-confidence MACS2 peaks passing the IDR threshold were retained for differential occupancy analysis. Finally, MEME-CentriMo was utilized to quantify motif centrality and spatial binding architectures. We integrated de novo ChIP-seq data with ATAC-seq and histone modification landscapes, ensuring all tracks were processed through a unified computational pipeline.
Results: Phenotypically, PPARD-overexpressing MDA-MB-231 represents a proliferative hybrid state, whereas PPARD-overexpressing HCC1937 exhibits a stem-like hybrid state. De novo motif discovery confirmed significant PPARD recruitment in both models but revealed a distinct topological shift. In MDA-MB-231, unique peaks displayed a centralized, unimodal distribution, indicating direct DNA binding at promoter-proximal regions. In contrast, HCC1937 unique peaks exhibited a bimodal motif architecture, suggesting indirect tethering at distal enhancers. Epigenetically, PPARD occupies an ""epigenetic braking"" zone (H3K27me3/H3K4me2) at the SNAI1 promoter in MDA-MB-231 to sustain proliferation, while in HCC1937, it coincides with ""Identity Peaks"" (H3K27ac) at GATA3 enhancers to favor stemness.
Conclusion: PPARD acts as a context-specific molecular switch that rewires the TNBC transcriptional landscape through topological reprogramming. By shifting between direct promoter binding and indirect enhancer tethering, PPARD differentially recruits epigenetic modifiers to key loci, orchestrating functional heterogeneity.

Co-authors: Peng Wang