Fig 1.
The lipogenic phenotype reprograms the epigenome in Ut-LMS.
Enhanced lipogenesis modifies the epigenome to transform mesenchymal cells in Ut-LMS. Akt, protein kinase B; mTORC1, mammalian target of rapamycin complex 1; FA, fatty acid; FASN, fatty acid synthase; SREBP-1c, sterol regulatory element-binding protein-1c; Ut-LMS, uterine leiomyosarcoma.
Fig 2.
FASN and palmitate modifies H3K methylation and acetylation in Ut-LMS.
(A-B) Immunoblot showing FASN expression in SK-LMS-1 and FASN- or EV-transduced SK-UT-1 and Ut-LMS CRC cells. FASN was introduced by retroviral vector and positive clones (#2, 5, 6, 8) were selected by puromycin. Clone (#5) with the highest FASN expression was amplified. Non-transformed Ut-LMS cell line CRC-2 was established from patient tissue, and expresses FASN at a high level. (B) Time-dependent H3K alterations mediated by forced FASN overexpression in SK-UT-1. (C) H3K alterations mediated by FASN siRNA knockdown in SK-LMS-1. Scramble or FASN siRNA were transfected into high FASN expressing SK-LMS-1 cells and lysates were harvested at indicated time points. (D) Palmitate reproduces H3K modification in dose dependent manner in low FASN expressing parental SK-UT-1 cells. Cells were treated with palmitate at indicated dose (μM) for 48hr, and cell lysates were harvested for histone 3 detection. The images are representative of at least three experiments. Cropped blots are from gels run under same condition. FASN, fatty acid synthase; EV, empty vector; Ut, uterine; LMS, leiomyosarcoma. CRC, conditionally reprogrammed cells.
Fig 3.
FASN alters H3K9 acetylation and methylation enzyme activities.
SK-UT-1-EV or–FASN cells were lysed, and nuclear extract was subjected to ELISA to measure the following histone modification enzyme activities: (A) Histone Acetylase (HAT); (B) Histone Deacetylase (HDAC); (C) Histone Methyltransferase (HMT); (D) Histone Demethylase (HDM). *, p<0.05 FASN-transduced SK-UT-1 vs. EV-transduced SK-UT-1 cells.
Fig 4.
FASN induces H3K9me3 to repress CRISP1 expression.
(A) ChIP-seq was performed in SK-UT-1-EV and–FASN cells with anti-H3K9me3 antibody. Enriched genomic regions were compared between SK-UT-1-FASN and SK-UT-1-EV. Screenshot shows CRISP1 is enriched for H3K9me3 in SK-UT-1-FASN cells (solid line) vs. SK-UT-1-EV (dot line); (B) Enrichment of CRISP1 binding to SK-UT-1-FASN genomic DNA by ChIP-PCR. Immunoprecipited chromatin DNA by H3K9me3 antibody was reversed crosslinking, purified and subject to q-PCR for CRISP1 gene. (C) qRT-PCR for CRISP1 expression. SK-UT-1-EV or–FASN cells were lysed for total RNA extraction, cDNA synthesis and q-PCR of CRISP1. *, p<0.05 FASN-transduced SK-UT-1 vs. EV-transduced SK-UT-1 cells. Data was normalized to GAPDH or β-actin. Results are expressed as the percentage of input DNA.
Fig 5.
FASN promotes Ut-LMS proliferation and increases Ut-LMS motion.
(A) DIMSCAN assay in SK-UT-1-EV and -FASN cells after 72 hrs incubation in 96 wells plate; (B) DIMSCAN assay in FASN siRNA-transfected SK-LMS-1 cells at 72 hr. Quantified cell motion detected by 72 hr time-lapse video microscopy. (C)Plastic plate; (D)Fibronectin-coated plate. Cell motion was recorded, tracked and quantitated by Image-Pro Premier 9.1.4. *, p<0.05 FASN-transduced SK-UT-1 vs. EV-transduced SK-UT-1 cells; +, p<0.05 FASN siRNA transfected SK-LMS-1 vs. scramble siRN transfected SK-LMS-1 cells.
Fig 6.
Scratch-wound assay of Ut-LMS.
(A) Migration of SK-UT-1-EV and SK-UT-1-FASN cells at 0 and 24 hr after introducing “wound.” (B) Migration of SK-LMS-1 cells at 24 (0 hr) and 48 (24 hr) hr after transfection with scrambled siRNA or siRNA targeting FASN (wound introduction).