Insights and modulation of RNA polymerase–dependent R-loop and dsRNA in Fanconi anemia hematopoietic stem cells
Michihiro Hashimoto, Xiaomin Feng, Jie Bai, Huimin Zeng, Tian Li, Jue Li, Terumasa Umemoto, Paul R. Andreassen, Gang Huang
Michihiro Hashimoto, Xiaomin Feng, Jie Bai, Huimin Zeng, Tian Li, Jue Li, Terumasa Umemoto, Paul R. Andreassen, Gang Huang
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Research Article Cell biology Hematology

Insights and modulation of RNA polymerase–dependent R-loop and dsRNA in Fanconi anemia hematopoietic stem cells

Abstract

Fanconi anemia (FA) is the most common BM failure (BMF) syndrome. FA genes have a role in suppressing DNA-RNA hybrids, termed R-loops, which can be generated via transcription mediated by RNA polymerase (RNAP). How these processes, including a role in fate determination of hematopoietic stem cells (HSCs), are related to BMF is largely unknown. Single FA gene KO in mice does not recapitulate most phenotypes observed in patients with FA. Thus, we generated a mouse model for FA by introducing heterozygous Setd2, which restricts RNAP-dependent transcription. We showed that FA patient–derived cells and Setd2+/– Fanca–/– HSCs share increased R-loop and dsRNA levels and a ribosomal biogenesis defect. Further, Setd2+/– Fanca–/– HSCs displayed cell cycle arrest, mitotic errors, and BMF phenotypes. Importantly, utilizing our Setd2+/– Fanca–/– mice, we discovered that Juglone, a pan-RNAP inhibitor, reduces R-loop and dsRNA and reverses ribosomal biogenesis defects and mitotic errors, thereby rescuing BMF. This study establishes a mouse model that underscores a key role for R-loop formation, ribosomal biogenesis defects, and mitotic errors in HSCs in driving BMF in FA. We also introduce a potential therapeutic avenue based upon pan-inhibition of RNAPs utilizing Juglone.

Authors

Michihiro Hashimoto, Xiaomin Feng, Jie Bai, Huimin Zeng, Tian Li, Jue Li, Terumasa Umemoto, Paul R. Andreassen, Gang Huang

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Figure 4

HSCs from Setd2+/– Fanca–/– mice display cell division defects.

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HSCs from Setd2+/– Fanca–/– mice display cell division defects. (A) Sche...
(A) Schematic of the experimental setup for B–D. (B and C) Proliferative capacity (B) and HSC [lineage (–), c-Kit (+), EPCR (+), CD48 (–), and CD150 (+)] frequency (C) of control (WT) or Setd2+/– Fanca–/– HSCs, determined as number of cells after 5 days of liquid culture relative to number at day 1, and HSCs as percentage of total at day 5, respectively. Cultures of HSCs were counted with Celigo in B and were analyzed by flow cytometry in C; for each condition, n = 3 in B and n = 4 in C. One-tailed Student’s t test. (D) Counts by hand of the number of cells in each well at 2 days after establishing single-cell cultures of control or Setd2+/– Fanca–/– HSCs. One-tailed Student’s t test; n = 3 for each condition. (E–H) Analysis comparing the cell division histories of control versus Setd2+/– Fanca–/– HSCs in 5-day liquid cultures. HSCs were sorted and stained with CytoTell green, then cultured for 5 days. At day 5, HSCs were stained with surface markers, and cell division history was analyzed with flow cytometry based on the CytoTell green signal in various peaks. Low division, medium division, and high division numbers were based on CytoTell green signal associated with 1–3, 4–8, and 9 or more divisions, respectively. One-tailed Student’s t test; n = 3 for each condition. (I) Visualization of nuclei in HSCs from control and Setd2+/– Fanca–/– mice, as a measure of mitotic errors, after 5 days in liquid culture. (J) Quantification of cells with abnormal nuclei (binucleated or micronucleated), indicating mitotic errors. One-tailed Student’s t test; n = 3 for each condition. (K) Schematic depicting nuclear morphologies and mitotic errors in controls and Setd2+/– Fanca–/– HSCs during cell proliferation. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.