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Methylated RNA immunoprecipitation sequencing in the Kasumi-1 cell line treated with CoCl2 and echinomycin

ABSTRACT: To evaluate the role of HIF1α in modulating the m6A abundance, we conducted m6A sequencing in the Kasumi-1 cell line treated with CoCl2 and echinomycin. CoCl2 can mediate the stabilization of HIF1α protein. Echinomycin is a small-molecule inhibitor of HIF1α, which functions through sequence-specific binding to HRE to competitively inhibit HIF1α binding to its target genes. The m6A sequencing results of the Kasumi-1 cell line treated with CoCl2 and non-treated control were uploaded previously (GSE168778). The differential m6A peaks analysis between CoCl2 + echinomycin group and CoCl2 group was performed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200605 | GEO | 2022/07/01

REPOSITORIES: GEO

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Project description:The Mexican axolotl provides a powerful model to investigate mechanisms of tissue regeneration. A recent chemical screen found that HDAC inhibitor romidepsin, administered for only 1-minute post amputation (1 MPA), blocks axolotl tail regeneration. Here, we tested the potential for cobalt chloride (CoCl2), a chemical stabilizer of HIF1a and inducer of hypoxia, to rescue romidepsin-inhibition of tail regeneration. Tail regeneration was partially rescued when embryos with amputated tails were co-treated with romidepsin and CoCl2. However, extending the CoCl2 dosage window either inhibited regeneration (CoCl2:0-30 MPA) or was lethal (CoCl2:0-24 hours post amputation; HPA). CoCl2:0-30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, and blocked regeneration. In contrast, CoCl2 treatment of non-amputated embryos or CoCl2:60-90 MPA treatment of amputated embryos did not affect wound healing or inhibit tail regeneration. To further investigate the contrasting effects of CoCl2, microarray analysis was performed to identify differentially expressed genes at 3 HPA. CoCl2-romidepsin:1 MPA treatment significantly increased the expression of transcription factors associated with appendage regeneration, while CoCl2:0-30 MPA significantly increased expression of hemoglobin and platelet-specific transcripts, consistent with hemorrhage and an impaired hemostatic response. Also, CoCl2:0-30 MPA significantly increased expression of hypoxia inducible genes, including genes that encode Hif1a interacting proteins and heat shock proteins (HSP); in contrast, genes encoding TGFB signaling components were significantly downregulated. Using additional chemical inhibitors of tail regeneration, we identified transcriptional responses associated with HSP90 activity and TGFB signaling. Notably, geldanamcin decreased transcription of matrix metalloproteinases and sustained muscle-specific gene expression, suggesting a role for HSP90 in regulating extracellular matrix remodeling and muscle dedifferentiation. Our study shows the power of using chemical tools to precisely identify temporal windows within which critical biological processes are enacted during tissue regeneration.

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Methylated RNA immunoprecipitation sequencing in the Kasumi-1 cell line treated with CoCl2 and echinomycin

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