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. 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.