A genome-wide shRNA screen to identify genes regulating ERα signalling and oestrogen-dependent proliferation in breast cancer cells

AbstractEpigenetics maintains cell-identity specific gene-expression patterns. However, within a population of isogenic cells of the same identity, a substantial variability in gene expression and responsiveness is still observed. Transcription bursting is a substantial source of this gene-expression variability or ‘noise’, contributing to phenotypic heterogeneity and potentially driving both physiological and pathological processes such as differentiation or tumorigenesis and drug resistance. Identification of transcription-bursting dynamics at a genome-wide scale has been restricted to inferring bursts in mRNA production computationally from the heterogeneity of mRNA levels in single cell transcriptomic data. Systematic characterisation of the genomic and epigenetic chromatin context of genes with defined transcription bursting behaviour has been incomplete. Here, we measured the bursting of transcription itself by genome-wide nascent RNA sequencing of breast cancer MCF-7 cells upon synchronisation of transcription with a transcription elongation inhibitor and by calibration using live cell imaging of nascent PP7-tagged GREB1 transcription. Comparing across the entire genome, we find transcription bursting to be ubiquitous, with burst sizes of up to 160 transcripts. Transcription bursting attributes ~85% to a trend in the variation in steady state gene expression between genes, whereas both burst frequency and nascent transcript degradation attribute minimally. Individual genes deviate strongly from this trend and engage both in anomalous burst size and frequency. We find that the presence of the TATA box or Inr sequence within gene promoters significantly predicts a larger burst size, as do promoter-associated YY1 and E2F1 transcription-factor binding motifs. Enrichment of the transcription start site with epigenetic marks such as H3K79me2 and H3Kl4ac is also strongly associated with the transcription burst size. Finally, we show that in these MCF-7 breast-cancer cells, genes with a larger transcription burst size exhibit a larger immediate transcriptional response following endocrine drug treatment. Our genome-wide transcription-bursting analysis method paves the way to elucidate the dynamic role of epigenetic regulation on dynamic transcription in pathophysiology.

Download Full-text

Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells

Oncogene ◽

10.1038/onc.2015.303 ◽

2015 ◽

Vol 35 (19) ◽

pp. 2465-2474 ◽

Cited By ~ 16

Author(s):

S T Workenhe ◽

T Ketela ◽

J Moffat ◽

B P Cuddington ◽

K L Mossman

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Oncolytic Virus ◽

Splicing Factor ◽

Virus Activity ◽

Genome Wide ◽

Shrna Screen

Download Full-text

A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells

Oncogene ◽

10.1038/sj.onc.1210712 ◽

2007 ◽

Vol 27 (7) ◽

pp. 1019-1032 ◽

Cited By ~ 143

Author(s):

C Williams ◽

K Edvardsson ◽

S A Lewandowski ◽

A Ström ◽

J-Å Gustafsson

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Estrogen Receptor Alpha ◽

Estrogen Receptor Beta ◽

Genome Wide ◽

A Genome ◽

Genome Wide Study ◽

Receptor Beta

Download Full-text

Abstract 5137: A genome wide RNAi screen identify STAT3 activation as essential for ErbB2 mediated transforamtion of breast cancer cells

10.1158/1538-7445.am2012-5137 ◽

2012 ◽

Author(s):

Ruth Rodriguez Barrueco

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Rnai Screen ◽

Genome Wide ◽

A Genome

Download Full-text

Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells

Antioxidants ◽

10.3390/antiox10081232 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1232

Author(s):

Sadaf Harandi-Zadeh ◽

Cayla Boycott ◽

Megan Beetch ◽

Tony Yang ◽

Benjamin J. E. Martin ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Dna Methyltransferase ◽

Epigenetic Marks ◽

Chip Sequencing ◽

Dietary Polyphenols ◽

A Genome ◽

Epigenetic Patterns

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 μM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Download Full-text

Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020478118 ◽

2021 ◽

Vol 118 (5) ◽

pp. e2020478118

Author(s):

Tobias Wijshake ◽

Zhongju Zou ◽

Beibei Chen ◽

Lin Zhong ◽

Guanghua Xiao ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Suppressor ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Target Genes ◽

Cellular Proliferation ◽

Beclin 1 ◽

Beta Catenin ◽

A Genome ◽

E Cadherin

Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1–specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin–mediated tumor suppression in breast cancer cells.

Download Full-text