RESUMO
GATA3 is known to be one of the most frequently mutated genes in breast cancer. More than 10% of breast tumors carry mutations in this gene. However, the functional consequence of GATA3 mutations is still largely unknown. Clinical data suggest that different types of GATA3 mutations may have distinct roles in breast cancer characterization. In this study, we have established three luminal breast cancer cell lines that stably express different truncation mutants (X308 splice site deletion, C321 frameshift, and A333 frameshift mutants) found in breast cancer patients. Transcriptome analysis identified common and distinct gene expression patterns in these GATA3 mutant cell lines. In particular, the impacts on epithelial-to-mesenchymal transition (EMT) related genes are similar across these mutant cell lines. Chromatin localization of the mutants is highly overlapped and exhibits non-canonical motif enrichment. Interestingly, the A333 frameshift mutant expressed cells displayed the most significant impact on the GATA3 binding compared to X308 splice site deletion and C321fs mutants expressed cells. Our results suggest the common and different roles of GATA3 truncation mutations during luminal breast cancer development.
RESUMO
The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.