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RE1 silencing transcription factor (REST) negatively regulates ALL1-fused from chromosome 1q (AF1q) gene transcription

BMC Molecular Biology (2015) 16(1)
DOI: 10.1186/s12867-015-0043-7
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Abstract

Background: ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive. Results: Our study showed that REST (RE1 silencing transcription factor), a key transcription factor in neurodevelopment, could down-regulate the gene expression of AF1q. The promoter assay identified a neuron-restrictive silencer element at -383 to -363 bp of human AF1q promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) confirmed the binding of REST to the NRSE in AF1q gene promoter. Additionally, the negative correlation between the expression levels of Af1q and Rest in mice neurodevelopment supported the negative regulation of AF1q by REST and the potential functions of AF1q in neurodevelopment. Conclusion: These results demonstrate that REST regulates AF1q gene transcription through directly binding to a NRSE at -383 to -363 bp of AF1q promoter.

Figures

  • Fig. 1 REST inhibited human AF1q gene transcription. a Real time PCR showed that REST expression was decreased by psiREST. SYBR Green gene expression assay of REST was performed. 18S rRNA was used as the internal control. The values represent the mean ± SE (n = 3). *P < 0.05 by Student’s t test. Con control. b RT-PCR showed that REST expression construct pREST increased REST mRNA expression, and REST knock down construct psiREST decreased REST mRNA expression. β-actin was used as the internal control. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test. c The knockdown effect of psiREST was verified in protein level by Western blot. REST was detected with anti-REST antibody. β-actin was used as the internal control. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test. d RT-PCR showed that AF1q mRNA was repressed by REST overexpression in HEK293 cells. β-actin was used as the internal control. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test. e Genomic organization of AF1q gene. AF1q gene had two exons and one intron. TSS, transcription start site. f pAF1q-promoter contains the functional promoter of AF1q gene, containing the 1809-bp fragment from the human AF1q gene 5′UTR, was transfected into HEK293 cells. +1 is the first base of the first exon. Dual-luciferase activity was measured by a luminometer 24 h after transfection. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test. g The expression of REST was examined by Western bolt. REST was detected with anti-REST antibody. Different mass ratios were marked under the Western blot figure. β-actin was used as the internal control. h AF1q gene promoter was inhibited by REST. REST was co-transfected with pAF1q promoter into HEK293 cells. Dual-luciferase activity was measured 24 h after transfection by a luminometer. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test
  • Fig. 2 Identification of the functional NRSE site in AF1q gene promoter. a Schematic diagrams of the AF1q promoter deletion constructs consisting of a 5′-flanking region with serial deletions cloned into the promoter-less vector plasmid pGL3-Basic in front of the luciferase reporter gene. Arrow indicated the direction of transcription. The numbers represented the end points of each construct. The positions of two putative NRSE sites were shown at the bottom. b The deletion plasmids were confirmed by sequencing and restriction enzyme digestion on a 1.5 % agarose gel. Vector size is 4.7 kb, and the AF1q gene 5′-flanking fragment inserts ranged from 0.6 to 1.8 kb. c HEK293 cells were co-transfected with REST expression vector and various AF1q promoter deletion constructs. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test. d pAF1qNRSEmut was made to contain the mutant NRSE site at −383 to −363 of p AF1qAluc, where (5′-TTAGCTGGGCGTGGTGGCGGA-3′) was mutated to (5′-TTAGCTGGaaGTctgGaaGGA-3′). e HEK293 cells were co-transfected with REST expression vector and AF1q promoter or pAF1qNRSEmut. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represented the mean ± SE (n = 3). *P < 0.01 by Student’s t test
  • Fig. 3 a EMSA was performed with IRDye 800-labeled NRSE site of AF1q promoter. Competition assays were performed by further adding different concentration of molar excess of unlabeled competitive oligonucleotides. b The addition of anti-REST antibody further shifted DNA–protein complex band. Lower panel showed a longer running time. c The anti-REST antibody actually immunoprecipitated REST protein. Lane 1 was input. Lane 2 was immunoprecipitates by anti-REST antibody. Lane 3 was immunoprecipitates by IgG antibody. And the lower bands were antibody heavy chain. d Anti-RNA polymerase II antibody was used to immunoprecipitate the GAPDH promoter region in CHIP assay in HEK293 cells. A pair of primers targeting GAPDH was used to amplify GAPDH. Signals amplified from input were used as size markers for PCR. IgG and H2O were used as negative controls. e Anti-REST antibody was used to immunoprecipitate the cross-linked REST-DNA complex in CHIP assay in HEK293 cells. A pair of primers targeting AF1q was used to amplify AF1q-REST. Signals amplified from input were used as size markers for PCR. IgG and H2O were used as negative controls. And anti-RNA Polymerase II antibody was used as positive control. Si-REST was from cells transfected with psiREST
  • Fig. 4 Af1q was negatively correlated with REST in mice neurodevelopment. a Quantitative RT-PCR was performed on cDNA templates prepared from normal mouse brain aging at embryonic days 13.5 (E13.5) and 18.5 (E18.5); at postnatal P1, P7, and P14; and in adult mice. One to three mice were used in each time point as indicated by the numbers after the hyphens. P < 0.05 by Spearman correlation test

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Hu, Y., Sun, Q., Zhang, C., Sha, Q., & Sun, X. (2015). RE1 silencing transcription factor (REST) negatively regulates ALL1-fused from chromosome 1q (AF1q) gene transcription. BMC Molecular Biology, 16(1). https://doi.org/10.1186/s12867-015-0043-7

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