Project description:To investigate the function of A3B, we established 92.1 cell lines with A3B knocked down and MEL290 cell lines with A3B overexpression. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cell lines with two replicates.
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations. Hormone-depleted MCF-7 breast cancer cell line was treated with estrogen (100 nM), H2O2 (10 mM) or vehicle for 45 minutes. H2AX ChIP-seq was performed using Illumina methodology.
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations.
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations.
Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B belongs to the APOBEC3 enzyme family, which consists seven closely related DNA deaminases that catalyse cytosine-to-uracil (C>U) editing of single-stranded DNA (ssDNA). Here, we adopted single-strand DNA protein-immunoprecipitation sequencing (SPI-seq) to capture the ssDNA bound by flag-tagged A3B protein. Quantification on sequence reads was carried out to investigate the effect of 17-beta estradiol (E2) on A3B binding to DNA.
Project description:To test if A3B has RNA editing activity we performed SNV calling from WES of 8 mouse livers (Samples 1-8) and 8 pancreas (Samples 7-16)
