Fig 2: FAS rs2234767 A and rs1800682 G alleles affect coupled SP1 and STAT1 recruitment to chromatin.(A) Chromatin immunoprecipitation (ChIP) of the FAS promoter with three different genotypes (rs2234767 GG/rs1800682 AA, rs2234767 GA/rs1800682 AG and rs2234767 AA/rs1800682 GG) using antibody for SP1 and STAT1(single pool generated from triplicate biological samples/manipulation; triplicate measurements/pool; mean ± SE). (B) Sequential ChIP of the FAS promoter containing SP1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). (C) Sequential ChIP of the FAS promoter containing STAT1 motif immunoprecipitated first using antibody for SP1 followed by antibody for STAT1 (upper) or first using antibody for STAT1 followed by antibody for SP1 (lower). For all combined genotypes in the figure, left genotype arises from the rs2234767 and right from the rs1800682 polymorphism.

Fig 3: Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. (a,b) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene (a) and DNMT1 gene (b). (c) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. (d) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. (e,f) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter (e) or DNMT1 promoter (f). IgG, control immunoglobulin. (g,h). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc (g) or DNMT1-luc (h) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.

Fig 4: SP1 transcriptional upregulated USP27X‐AS1 expression. (A) Possible transcription factors of USP27X‐AS1 predicted by online tools. (B) Analyses for the expression correlations between SP1 and USP27X‐AS1 in TCGA‐LIHC; r = .3477, p < .001. (C) Analyses for the expression correlations between FOXA2 and USP27X‐AS1 in TCGA‐LIHC; r = .1325, p = .01. (D) Analyses for the expression correlations between ZNF263 and USP27X‐AS1 in TCGA‐LIHC; r = .1516, p = .0037. (E) Changes of SP1 and USP27X‐AS1 RNA level after SP1 expression was interfered in MHCC97H. (F) Changes of AKT protein level after SP1 expression was interfered in MHCC97H. (G) Changes of SP1 and USP27X‐AS1 RNA level after SP1 overexpression in HLF. (H) Changes of AKT protein level after SP1 overexpression in HLF. (I) Schematic diagram of the deletion and selective mutation of USP27X‐AS1 promoter. (J and K) Analysis identified SP1‐responsive regions in the USP27X‐AS1 promoter. (L and M) CUT–RUN analysis of SP1 binding to the USP27X‐AS1 promoter in MHCC97H (L) and HLF (M) cells. Data and error bars are shown as mean ± SD of triplicate independent replicate experiments. *p < .05, **p < .01, ***p < .001, ns: no significance. Data were analysed by paired Student's t‐test (B–E, G and J–M).

Fig 5: Identification of SP1 as the mutual transcription factor of MRPs and 12LOX. a Bioinformatic analysis of transcription factors of MRP1, MRP4, Snail, Slug, 12LOX, COX1, and CYP2J2. b SP1 binding sequences in the promoters of MRP1 (ABCC1), MRP4 (ABCC4), and 12LOX (ALOX12). c ChIP assay of SP1 binding on the promoters of MRP1 (ABCC1), MRP4 (ABCC4), and 12LOX (ALOX12). d, e The mRNA (d) and protein (e) expression levels of SP1 in SKOV3 and SKOV3-R cells. Data in (d) are expressed as mean ± SEM of 4 independent experiments. ***P < 0.001. f The unpaired Student’s t test was used to analyze the expression of SP1 in the GEO database (GSE114206). Data are expressed as mean ± SEM and *P < 0.05. g Patient survival data were obtained from TCGA database and overall survival probability was then calculated using the Kaplan-Meier method and the differences in survival curves were analyzed using the log-rank test. P-values of 0.05 or less were considered significant