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An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles
Published on: March 18, 2015
Doxorubicin, a DNA intercalator, inhibits transcription elongation
Mathew Tempel1, Kari Green1, Dhanvi Prajapati1
1Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 3P4, Canada.
Abstract:
Doxorubicin is a chemotherapeutic drug for cancer that intercalates into nucleosome-free regions at promoters. Doxorubicin was reported to result in loss of histone H3 trimethylated lysine 4 (H3K4me3). To further explore doxorubicin's mechanism of action, we determined the genomic location of the binding sites of doxorubicin in leukemic cells. The effect of doxorubicin intercalation into the chromatin of leukemic cells on histone modifications was also determined. We show that doxorubicin binding sites were present in the nucleosome-free regions associated with regulatory regions (promoters, enhancers, and super-enhancers) and in the gene body (introns). Doxorubicin treatment did not alter the levels of H3K4me3 and many other histone modifications but significantly lowered H2B ubiquitinated at lysine 120 (H2BK120ub), an elongation-dependent modification. Lastly, we demonstrate that doxorubicin results in the degradation of the largest subunit (RPB1) of RNA polymerase II.
Insights
Doxorubicin chemotherapy drug binds to DNA in cancer cells, affecting gene regulation. It degrades RNA polymerase II, impacting cancer cell function.
Area of Science:
- Oncology
- Molecular Biology
- Genomics
Background:
- Doxorubicin is a widely used chemotherapy agent.
- Its mechanism involves DNA intercalation, primarily at promoter regions.
- Previous studies suggested it affects histone modifications like H3K4me3.
Purpose of the Study:
- To map doxorubicin's genomic binding sites in leukemic cells.
- To investigate doxorubicin's impact on histone modifications.
- To elucidate the drug's precise mechanism of action.
Main Methods:
- Chromatin immunoprecipitation (ChIP) to identify doxorubicin binding sites.
- Analysis of histone modifications using ChIP.
- Western blotting to assess protein levels, including RNA polymerase II.
Main Results:
- Doxorubicin binds to nucleosome-free regions in regulatory elements and gene bodies.
- It does not alter H3K4me3 levels but reduces H2B ubiquitination (H2BK120ub).
- Doxorubicin treatment leads to the degradation of RNA polymerase II subunit RPB1.
Conclusions:
- Doxorubicin targets specific genomic regions in leukemic cells.
- Its mechanism involves downregulating H2BK120ub and degrading RPB1.
- These findings provide new insights into doxorubicin's anti-cancer effects.
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