PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity

Abstract

Chromatin remodeling and N⁶-methyladenosine (m⁶A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m⁶A methylation of poly(A)⁺ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m⁶A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m⁶A methylation was located at A1881. The level of m⁶A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m⁶A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

Keywords: DNA damage response; PARP1; chromatin accessibility; m(6)A methylation; radiosensitivity.

Graphical abstract

Figure 1

Decreased m⁶A methylation of poly(A)⁺ RNA upon exposure to IR (A) The m⁶A modification level of total poly(A)⁺ RNA in HeLa cells was detected by RNA methylation quantification (colorimetric assay) at given times after 4-Gy γ-ray irradiation. (B and C) m⁶A methylation level of total poly(A)⁺ RNA in HeLa cells was detected by m⁶A dot blot assay (B) and the corresponding quantification (C) at given times after 4-Gy γ-ray irradiation. (D and E) The m⁶A methylation level of total poly(A)⁺ RNA in HeLa cells was detected 2 h after different doses of γ-ray irradiation by m⁶A dot blot assay (D) and corresponding quantification (E). (F) The m⁶A methylation level of total poly(A)⁺ RNA in HeLa cells was detected 2 h after doses of γ-ray irradiation by RNA methylation quantification (colorimetric assay). (G and H) The expression of METTL3 was detected by qPCR 2 h after 4-Gy irradiation (G) or western blot analysis (H) at given times after 4-Gy γ-ray irradiation. (I and J) Effect of transfecting/overexpressing exogenous METTL3 vectors on the change in m⁶A level of total poly(A)⁺ RNA at 2 h after 4-Gy irradiation was detected by m⁶A dot blot assay (I) and corresponding quantification (J) in HeLa cells. All data represent the mean ± SD from three independent experiments. Compared with the control, ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Suppression of METTL3 expression by irradiation and PARP1 inhibition (A) Decreased binding of the transcription factors NFIC and TBP to the METTL3 promoter after 4-Gy irradiation, as detected by ChIP-qPCR assay. (B) Confirmation of decreased binding of PARP1 to the METTL3 promoter in HeLa cells after 4-Gy irradiation, determined by ChIP-qPCR assay. (C) HEK293T cells were transfected with the indicated combination of expression constructs for 24 h and incubated with purified proteins, followed by pull-down and immunoblotting with biotin beads. (D–G) Inhibition of METTL3 mRNA (D and F) and protein (E and G) expression by the PARPis NMS-P118 and olaparib (D and E) or knockdown of PARP1 (F and G). (H–K) Observation of the combined effect of radiation and PARPis (H and I) or knocking down PARP1 and rescuing PARP1 (J and K) on the expression of METTL3 mRNA (H and J) and protein (I and K). All data represent the mean ± SD from three independent experiments. Compared with the control, ∗p < 0.05, ∗∗p < 0.01.

Figure 3

Radiation and PARP1 inhibition suppress the accessibility of METTL3 promoter chromatin (A and B) The effects of radiation and PARP1 on the openness of METTL3 promoter chromatin, as detected by MNase digestion-qPCR assay. Cells were treated with 4-Gy γ-rays (A) or the PARPi NMS-P118. The chromatin DNA was digested with MNase and then subjected to qPCR assay of METTL3 promoter sequences. (C) The effect of the PARPi NMS-P118 on access of the transcription factors TBP and NFIC to METTL3 promoter chromatin, as detected by a TF-driven ChIP-qPCR assay. (D and E) The effects of depressing TBP and NFIC, and PARP1 on expression of METTL3 mRNA (D) and protein (E). (F and G) TBP/NFIC inhibition attenuated the increase in METTL3 mRNA (F) and protein (G) expression induced by PARP1 overexpression. (H and I) TBP/NFIC inhibition abrogated the rescue effect of overexpressed PARP1 on radiation-depressed METTL3 RNA (H) and protein (I) expression. All data represent the mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.

Figure 4

Depression of PARP1 attenuates m⁶A methylation of total poly(A)⁺ RNA and identification of potential m⁶A targets related to radiation-induced DNA damage (A and B) PARP1 depression resulted in reduced m⁶A modification of poly(A)⁺ RNA in HeLa cells as detected by m⁶A dot blot assay and quantification. (C and D) The effect of knockdown of PARP1 and METTL3 on m⁶A modification levels was detected by m⁶A dot blot assay and quantification. (E and F) The combined effects of radiation and PARPi on m⁶A modification levels as detected by m⁶A dot blot assay and quantification. (G and H) siRNA-depressed METTL3 abrogated the increased m⁶A modification induced by overexpressed PARP1 as detected by m⁶A dot blot assay and quantification. All data represent the mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01. (I) Sequence logo representing the consensus m⁶A motifs relative to differential m⁶A-modified loci identified in 4-Gy γ-ray-irradiated HeLa cells compared with unirradiated cells. Coexpression analysis was performed on transcriptome-wide m⁶A-seq profiling and RNA-seq profiling. (J) Histogram showing the altering trends of RNA expression and m⁶A modification pattern of genes in HeLa cells 2 h after irradiation compared with unirradiated cells.

Figure 5

Radiation and PARP1 inhibition depressed LPAR5 expression by modulating METTL3-mediated m⁶A modification and promoting decay of LPAR5 mRNA (A) Knockdown of METTL3 reduced the expression of LPAR5 protein. (B) Overexpressing METTL3 increased the expression of LPAR5 protein. (C) Radiation exposure led to depression of LPAR5 protein expression, which was rescued by overexpressing METTL3. (D and E) PARPis depressed the expression of LPAR5 protein (D), which was rescued by overexpressing METTL3 (E). (F) LPAR5 m⁶A methylation was reduced upon irradiation (F), which could be rescued by overexpressing METTL3. (G and H) LPAR5 m⁶A methylation was reduced upon knockdown of PARP1 (G) or METTL3 (H). (I) Overexpressing PARP1 enhanced LPAR5 m⁶A methylation. (J) The half-life of LPAR5 mRNA was shortened by knocking down METTL3 in HeLa cells. (K and L) In HeLa cells, METTL3 knockdown reduced the activity (K) and m⁶A methylation (L) of LAPR5 3′ UTR WT, and the A1881C mutation led to reduced activity but was no longer affected by METTL3 knockdown. (M) The half-life of LPAR5 WT or A1881C mutant was detected with or without METTL3 knockdown. All data represent the mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.

Figure 6

Irradiation promotes apoptosis and inhibits cancer cell proliferation through the PARP1-METTL3-m⁶A-LPAR5 axis (A–C) overexpressing METTL3 attenuated radiation-induced proliferation inhibition (A and B) and apoptosis induction (C) in HeLa and A549 cells. (D and E) Apoptosis induced by PARP1 depression (D), PARPi, or irradiation or combined treatment of IR and PARPi (E) was partially rescued by overexpressing METTL3. (F) LPAR5 knockdown induced apoptosis in HeLa and A549 cells. (G and H) Increased occurrence of apoptosis by knocking down METTL3 (G) or irradiation (H) was attenuated by overexpressing LPAR5-WT or LPAR5-A1881C but to a lesser extent by LPAR5-A1881C. (I) Apoptosis induced by radiation was attenuated by overexpressing METTL3 but not by overexpressing METTL3 and simultaneously knocking down LPAR5. (J and K) Verification of radiation-induced apoptosis via the PARP1-METTL3-LPAR5 axis. (J) Effects of LPAR5 knockdown, PARP1 overexpression, or METTL3 knockdown on apoptosis in irradiated HeLa cells and A549 cells. (K) Effects of overexpressing LPAR5, METTL3, NMS-P118 treatment or combination treatment on apoptosis induction in irradiated HeLa cells and A549 cells. All data represent the mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01.

Figure 7

Targeted inhibition of METTL3 suppresses tumor growth and exhibits a synergistic effect with radiation in vivo (A) Representative images of immunohistochemistry (IHC) analysis of PARP1, METTL3, and LPAR5 in a tumor tissue array of uterine cervix adenocarcinoma. The tissue array was composed of human cervical cancers and paired para-tumor tissues. (B–D) Quantitative comparisons between cervical cancers and paired para-tumor tissues for PARP1, METTL3, and LPAR5 expression IHC analyses. (E) Correlation analysis of METTL3 and LPAR5 expression in cervical cancers. (F–H) Tumor suppression by a small-molecule inhibitor of METTL3 and synergistic effect with radiation. (F) The tumor sizes in each group were monitored every 3 days starting from 1 day after radiation treatment. (G and H) Tumor sizes and weights in each group 21 days after radiation treatment. n = 7, ∗p < 0.05, ∗∗p < 0.01.