FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer [ChIP-seq]

The transcription factor FOXA1, which functions in epigenetic reprogramming, plays a critical role in breast cancer progression. However, the molecular mechanisms by which FOXA1 achieves its oncogenic functions remain elusive. Here, we demonstrate that the O-GlcNAcylation of FOXA1 increases breast cancer metastasis capacity by orchestrating the transcription of numerous metastasis regulators. O-GlcNAcylation at Thr432, Ser441 and Ser443 regulates the stability of FOXA1 and promotes its assembly with chromatin in breast cancer cells. We provide evidence that O-GlcNAcylation shapes the FOXA1 interactome, especially triggering the recruitment of the transcriptional repressor MECP2 and consequently stimulating FOXA1 chromatin-binding sites to switch to chromatin loci of adhesion-related genes, including EPB41L3 and COL9A2. Site-specific depletion of O-GlcNAcylation on FOXA1 affects the expression of various downstream genes and thus inhibits breast cancer proliferation and metastasis both in vitro and in vivo. Overall design: We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-HA magnetic beads in HA-FOXA1-WT or HA-FOXA1-3A stably expressed MCF-7 FOXA1 KO cells. MECP2 ChIP-seq was performed using anti-MECP2 antibody in the above cells. Next, MECP2 was knockdown by shRNA in FOXA1 rescued MCF-7 FOXA1 KO cells. HA-FOXA1-WT and HA-FOXA1-3A ChIP-seq were performed using anti-HA magnetic beads in MECP2 knockdown cells. Next-generation sequencing libraries were generated and amplified for 15 cycles. 100-300 bp DNA fragments were gel-purified and sequenced with an Illumina NovaSeq 6000 instrument by Novogene Technology (Beijing, China). Two biological replicates of each ChIP-seq were performed. We also analyzed gene expression in FOXA1-WT and FOXA1-3A expressed cells by RNA-seq.