Journal: bioRxiv

Article Title: Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

doi: 10.1101/2020.04.08.032169

Figure Lengend Snippet: (a) Principal component analysis of RNA-seq data displays the relationships between WT(blue), Y537S(yellow), and D538G(red) T-47D clones. (b) Heatmap shows expression levels for ligand-independent, mutant-specific shared, and allele-specific genes. (c) qPCR validation of ligand-independent ( CISH ) and mutant-specific ( CASC5 ) expression. Error bars indicate average ± SEM for two clones for each genotype and each treatment. Student’s two sample t-test was used: ** p<0.01, *** p<0.001, **** p<0.0001, and n.s. = not significant. (d) Enriched gene ontology terms for Y537S- and D538G-specific differentially regulated genes with Fisher’s exact test p-values are shown.

Article Snippet: Hormone-depleted media consisted of phenol-red free MEM (Thermo Fisher Scientific, 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin, and NEAA and insulin as described above for MCF-7 cells and phenol-red free RPMI (Thermo Fisher Scientific), 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin for T-47D cells.

Techniques: RNA Sequencing Assay, Clone Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

doi: 10.1101/2020.04.08.032169

Figure Lengend Snippet: (a) Principle component analysis of RNA-seq data captures the relationships between T-47D ER mutant clones and WT clones with short- or long-term E2 treatment. (b) Heatmap shows all mutant-specific genes separated by long-term E2-treatment expression (top: genes regulated by long-term E2 treatment; bottom: genes not regulated by long-term E2 treatment). (c) Percent of mutant-specific genes regulated by long-term ER activation in WT clones is shown. (d) Bar graphs show examples of genes that are regulated by long-term E2 treatment ( TBCD ) and that are differentially regulated by mutation only ( COPS2 ). (e) Significantly enriched gene ontology terms are displayed for Y537S-specific and D538G-specific gene sets partitioned based on overlap with long-term E2 regulated genes.

Article Snippet: Hormone-depleted media consisted of phenol-red free MEM (Thermo Fisher Scientific, 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin, and NEAA and insulin as described above for MCF-7 cells and phenol-red free RPMI (Thermo Fisher Scientific), 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin for T-47D cells.

Techniques: RNA Sequencing Assay, Mutagenesis, Clone Assay, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

doi: 10.1101/2020.04.08.032169

Figure Lengend Snippet: (a) Heatmap shows the binding signal of constant and mutant-specific ERBS in T-47D WT and mutant clones with addition of DMSO or E2. (b) Example locus of ligand-independent (constant) ER-binding, arrow indicates TSS of GREB1 . All tracks are normalized to the same scale. GREB1 ligand-independent gene expression is shown on the right. (c) Heatmap displays the signal of mutant-enriched or depleted sites only. (d) Example locus of mutant-enriched ER-binding. All tracks are normalized to the same scale. ULK2 gene expression is shown on the right. (e) Distance from mutant-specific genes to mutant-enriched and -depleted ERBS is shown as a cumulative distribution.

Article Snippet: Hormone-depleted media consisted of phenol-red free MEM (Thermo Fisher Scientific, 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin, and NEAA and insulin as described above for MCF-7 cells and phenol-red free RPMI (Thermo Fisher Scientific), 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin for T-47D cells.

Techniques: Binding Assay, Mutagenesis, Clone Assay, Expressing

Journal: bioRxiv

Article Title: Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

doi: 10.1101/2020.04.08.032169

Figure Lengend Snippet: (a) Heatmap displays ATAC-seq signal of T-47D mutant-enriched and -depleted chromatin accessibility sites. (b) Example locus of shared mutant-enriched accessible chromatin region is shown. Arrow indicates the TSS of CEP78 . All tracks are normalized to the same scale. CEP78 gene expression is shown on the right. (c) Cumulative distribution graphs show distance from Y537S mutant-specific genes to Y537S mutant-enriched accessible chromatin. (d) Percent of mutant-enriched or -depleted accessible chromatin regions that also exhibit ER binding is shown.

Article Snippet: Hormone-depleted media consisted of phenol-red free MEM (Thermo Fisher Scientific, 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin, and NEAA and insulin as described above for MCF-7 cells and phenol-red free RPMI (Thermo Fisher Scientific), 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin for T-47D cells.

Techniques: Mutagenesis, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

doi: 10.1101/2020.04.08.032169

Figure Lengend Snippet: (a) Enriched transcription factor DNA binding motifs are found at T-47D mutant-enriched accessible chromatin. (b) Gene expression is shown for CTCF , FOX family genes, and POU family genes. Bars represent RNA-seq normalized counts ± SEM for two clones for each genotype and each treatment. (c) Heatmaps display the intensity (depth) of accessible chromatin (red) or DNA-binding of three factors: OCT1 (yellow), CTCF (green), and FOXA1 (blue) at mutant-enriched accessible chromatin regions. qPCR analysis of mutant-specific genes is shown after 72 hours of siRNA-mediated knockdown of OCT1 (d) or CTCF (e). Bars represent average expression ± SEM of two replicates for two clones for each genotype and each treatment. Student’s two sample t-test was used: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, and n.s. = not significant.

Article Snippet: Hormone-depleted media consisted of phenol-red free MEM (Thermo Fisher Scientific, 5% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin, and NEAA and insulin as described above for MCF-7 cells and phenol-red free RPMI (Thermo Fisher Scientific), 10% charcoal-stripped fetal bovine serum, and 1% penicillin-streptomycin for T-47D cells.

Techniques: Binding Assay, Mutagenesis, Expressing, RNA Sequencing Assay, Clone Assay