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Searching for the fusion transcripts created during proteotoxic stress

ABSTRACT: Searching for the fusion transcripts created during proteotoxic stress

PROVIDER: PRJEB73803 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR12737079_1.fastq.gz	Fastqsanger.gz
	ERR12737079_2.fastq.gz	Fastqsanger.gz
	ERR12737080_1.fastq.gz	Fastqsanger.gz
	ERR12737080_2.fastq.gz	Fastqsanger.gz
	ERR12737081_1.fastq.gz	Fastqsanger.gz

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Searching for the fusion transcripts created during proteotoxic stress

Project description:To verify the hypothesis that proteotoxic stress induces the formation of fusion transcripts in non-cancerous and cancerous cells derived from breast epithelium, we exposed the cells to heat shock (HS, 1h at 43 degrees). We selected MCF10A non-tumorigenic breast epithelial cells (isolated from the mammary gland with fibrocystic disease) and MCF7 breast adenocarcinoma cells (estrogen receptor-positive, ER+). Global transcriptome analysis was performed in untreated cells and at 2h and 4h after HS. The TimeLapse-Seq method was used, which allows for obtaining information about newly produced RNA in relation to total RNA in one sequencing reaction [1]. To metabolically label the newly produced RNA, 4-thiouridine (4sU, a thymidine analog) was added to the medium for the duration of the experiment. To convert 4sU to cytidine analogs (providing U-to-C mutations that enable tagging of new transcripts when sequenced), samples were treated with 2,2,2-trifluoroethylamine (TFEA) and sodium periodate (NaIO4) as described in [1]. All libraries were sequenced on the Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 200M reads/sample. Obtained data were subjected to the standard RNA-seq data analysis procedure, including the additional steps required for TimeLapse-Seq, described in [1]. Identification of gene fusions was performed using the Arriba tool [2]. [1] Schofield JA. et al. Nat Methods. 2018, 15:221-225. [2] Haas BJ. et al. Genome Biol. 2019, 20:213.

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Project description:A novel oligonucleotide microarray design is described whereby one can screen for all known oncogenic fusion transcripts by one microarray hybridization. Measurements of chimeric transcript junctions are combined with exon-wise measurements of individual fusion partners. Keywords: Nimblegen custom-design The pilot data included a design with 68,861 oligonucleotide probes covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG) from six positive controls consisting of leukemia cell lines and prostate cancer biopsies.

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Project description:Leiomyosarcoma (LMS) is an aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We found that the expression of the heat shock proteins (HSP) gene family is upregulated with higher efficiency in normal cells. In contrast, upregulation of BH3-only proteins is higher in LMS cells. HSF1, the master regulator of HSP transcription, is sequestered into transcriptionally incompetent nuclear foci only in LMS cells, which explains the lower HSP upregulation. We also found that several compounds can enhance the cell death response to proteotoxic stress. Specifically, when low doses were used, an inhibitor of salt-inducible kinases (SIKs) and the inhibitor of IRE1, a key element of the unfolded protein response (UPR), support proteotoxic-induced cell death with strength in LMS cells and without effects on the survival of normal cells. Overall, our data provide an explanation for the higher susceptibility of LMS cells to proteotoxic stress and suggest a potential option for co-treatment strategies.

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