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Synthetic circuits for tracking human cell fate

ABSTRACT: Cells respond heterogeneously to DNA damage. We engineered genetic circuits to detect differential responses in a population that persist for many days post-stimulus. We used microarrays to compare memory and non-memory subpopulations 3 days after DNA damage or doxycycline exposure. MD12/p53R2-RE and MD10/TetOx2 cells were either exposed to UV (10uJ/m^2) or doxycycline (1 ug/mL, 24 hours) and allowed to recover 3 days before sortng of memory and non-memory cells and RNA extraction. Two replicates were submitted for each condition (UV memory, UV non-memory, dox memory, dox non-memory)

ORGANISM(S): Homo sapiens

SUBMITTER: Mara Inniss

PROVIDER: E-GEOD-38189 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Synthetic circuits for tracking human cell fate

Project description:Cells respond heterogeneously to DNA damage. We engineered genetic circuits to detect differential responses in a population that persist for many days post-stimulus. We used microarrays to compare memory and non-memory subpopulations 3 days after DNA damage or doxycycline exposure.

2012-05-24 | GSE38189 | GEO

Dynamic Maps of UV Damage Formation and Repair for the Human Genome

Project description:UV-induced DNA damage is the major carcinogen of skin cancer. We developed HS-Damage-seq to map UV damage with unprecedented sensitivity and resolution in human cells. While the distribution of UV-induced damage are mainly determined by sequence context and essentially uniform throughout the genome, transcription factors can affect damage formation at specific positions. With time-course experiments, we observed changes in damage distribution at particular genomic loci and chromatin states, which reflects the nucleotide excision repair preferences and complements the repair maps by XR-seq. Our results demonstrated that while initial damage is uniformly distributed, they are heterogeneously repaired throughout the genome. The combination of damage and repair maps provides a holistic perspective of UV damage and repair in the human genome.

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Project description:To characterize the mitochondrial stress response induced by Doxycycline in vivo in liver and kidneys, we administered doxycycline (Dox) at 500 mg/kg/day (mpkd) in the drinking water to 9 weeks-old germ-free C57BL/6J mice for 16 days, hence eliminating the potential confounding impacts of Doxycycline on the microbiome.

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Expression data from YAP-S127A Villin-rtTA crypts

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Analysis of oncogenic Kras-induced changes in the RNA-bound proteome of PDAC cells

Project description:In order to reveal the impact of oncogenic KRAS signaling on the RNA-bound proteome of Pancreatic Ductal Adenocarcinoma (PDAC), we carried out quantitative whole Transcriptome RNA Interactome Capture (RIC) analysis in tumour cells from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). For RIC, we employed Orthogonal Organic Phase Separation (OOPS)(Queiroz et al., 2019). In this method, UV-C crosslinking coupled with phenol-chloroform based phase separation is used to isolate RNA-crosslinked proteins, which concentrate in the interface fraction, whilst free proteins concentrate in the organic fraction. SILAC was employed for quantitative analysis of Dox induced (24hrs) changes in the RNA-bound proteome. To assess the impact of ERK signalling, an independent experiment was carried out in which Trametinib (10nM) was added to the cells at the same time as Dox (both with or without Dox cells). A total of 6 biological replicates without Trametinib, and 4 biological replicates with Trametinib, were conducted, with switching the Heavy and Light SILAC labels for +Dox and -Dox conditions. Both the Interface as well as the organic phase fractions were analysed by mass spectrometry. In a separate experiment, the total proteome changes in response to dox (24hrs) were quantified from two reciprocally SILAC labelled mixes of heavy and light iKras PDAC whole cell lysates (See the READ ME.XLS for details of the experimental conditions).

2023-02-12 | PXD030825 | Pride

CPSF-160 iCLIP

Project description:We performed iCLIP of CPSF-160- GFP as CPSF160 was believed to be the main RNA-binding subunit of CPSF. To this end, we used a stable HeLa cell line that expresses GFP-CPSF160 in a doxycycline (Dox)-dependent manner. After GFP-CPSF160 was induced, we irradiated the cells with UV-C (wavelength=254nm) to induce protein-RNA crosslinks and immunoprecipitated the complex with a GFP antibody.

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