TGF-β1 promotes CpG methylation in the intron 8 of Myo1f locus to inhibit SPI1 binding. (A) Correlation analysis of SPI1 and MYO1F/MYO1E, using expression datasets from the TCGA database via the GEPIA2 website. (B)Myo1f mRNA levels were detected by RT-qPCR from cultured neutrophils 48 h after transfection of siSpi1 (N = 3). (C) MYO1F protein levels were analyzed 72 h after transfection of siSpi1. (D) Murine SPI1-CHIP-Seq peak plot on chr17: 33771681–33826738. References are described in the main text. (E) FACS analyses of MYO1F on immune cells from WT mice (N = 5). (F) The transcription activity of 400-bp segment included each peak, measured by using a pGL3 vector luciferase reporter gene assay, after transfection into cultured neutrophils. (G) Schematic of SPI1-binding segment located on eighth intron. (H) The transcription activity of full-length and SPI1-binding truncated eighth intron measured by using a pGL3 vector luciferase reporter gene assay after 48 h of transfection into cultured neutrophils. (I) The transcription activity of 400-bp SPI1-binding segment 36 h after TGF-β1 5 ng/ml treatment. (J) ChIP-RT-qPCR on enriched DNA IP by anti-SPI1 pulldown; IgG was used as control. (K) Top: Schematic of CpG methylation distributed in eighth intron of chr17: 33799270–3379914. Bottom: Evaluation of CpG methylation by BSP sequencing. (L) The transcription activity of WT (CpG#2 mutant) and adenine-replaced mutant (CpG #2 mutant) of 400-bp SPI1-binding segment, 48 h after TGF-β1 5 ng/ml treatment. Data in B, C, E, F, and H–L represent one experiment of three independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (E, F, H–J, and L); two-tailed unpaired Student’s t test (B), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance. Neu, neutrophil. Source data are available for this figure: SourceData F7.
Figure 7.

TGF-β1 promotes CpG methylation in the intron 8 of Myo1f locus to inhibit SPI1 binding. (A) Correlation analysis of SPI1 and MYO1F/MYO1E, using expression datasets from the TCGA database via the GEPIA2 website. (B)Myo1f mRNA levels were detected by RT-qPCR from cultured neutrophils 48 h after transfection of siSpi1 (N = 3). (C) MYO1F protein levels were analyzed 72 h after transfection of siSpi1. (D) Murine SPI1-CHIP-Seq peak plot on chr17: 33771681–33826738. References are described in the main text. (E) FACS analyses of MYO1F on immune cells from WT mice (N = 5). (F) The transcription activity of 400-bp segment included each peak, measured by using a pGL3 vector luciferase reporter gene assay, after transfection into cultured neutrophils. (G) Schematic of SPI1-binding segment located on eighth intron. (H) The transcription activity of full-length and SPI1-binding truncated eighth intron measured by using a pGL3 vector luciferase reporter gene assay after 48 h of transfection into cultured neutrophils. (I) The transcription activity of 400-bp SPI1-binding segment 36 h after TGF-β1 5 ng/ml treatment. (J) ChIP-RT-qPCR on enriched DNA IP by anti-SPI1 pulldown; IgG was used as control. (K) Top: Schematic of CpG methylation distributed in eighth intron of chr17: 33799270–3379914. Bottom: Evaluation of CpG methylation by BSP sequencing. (L) The transcription activity of WT (CpG#2 mutant) and adenine-replaced mutant (CpG #2 mutant) of 400-bp SPI1-binding segment, 48 h after TGF-β1 5 ng/ml treatment. Data in B, C, E, F, and H–L represent one experiment of three independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (E, F, H–J, and L); two-tailed unpaired Student’s t test (B), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance. Neu, neutrophil. Source data are available for this figure: SourceData F7.

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