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, 385 (Pt 2), 557-64

Transcriptional Regulation of the Human DNA Methyltransferase 3A and 3B Genes by Sp3 and Sp1 Zinc Finger Proteins

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Transcriptional Regulation of the Human DNA Methyltransferase 3A and 3B Genes by Sp3 and Sp1 Zinc Finger Proteins

Artit Jinawath et al. Biochem J.

Abstract

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

Figures

Figure 1
Figure 1. Mithramycin A inhibits DNMT3A and DNMT3B promoter activities and mRNA expression
(A) Schematic structure of the 5′-region of the human DNMT3A and DNMT3B mRNAs. Boxed numbers indicate exons, and arrows indicate the positions of sense and antisense primers used for semiquantitative RT–PCR. Three distinct 1st exons of DNMT3A (1A, 1B and 1C) are driven by separate promoters (1st, 2nd and 3rd promoters respectively), and spliced to the common exon 2. The 5′-region of DNMT3B mRNA contains two alternative 1st exons (1A and 1B), which are spliced to a common exon 2. The structure of the novel alternative spliced variant of DNMT3B that lacks exon 5 is shown in the smaller Figure below. (B) The reporter construct containing DNMT3A promoters (1st and 2nd promoters, pGL3A-P1+2; 3rd promoter, pGL3A-P3) and DNMT3B promoters (1st promoter, pGL3B-P1; 2nd promoter, pGL3B-P2) were transfected into HEK-293T cells. Then, luciferase activity was determined in the absence or presence of 100 nM mithramycin A and expressed relative to the activity in the absence of mithramycin A. Results are expressed as the means±S.D. for three experiments. *P<0.05 with the paired t test. (C) Semiquantitative RT–PCR analysis of DNMT3A and DNMT3B 1st exon mRNA variants in HEK-293T cells after incubation with 100 and 200 nM mithramycin A.
Figure 2
Figure 2. DNMT3A and DNMT3B expression levels in Sp1- or Sp3-overexpressing cells
(A) Total RNA from HEK-293T cells transfected with Sp1, Sp3 or pRc/CMV were amplified by semiquantitative RT–PCR method using specific primers for the 3′-portions of DNMT3A and DNMT3B and all alternative 1st exons of each gene. The experiments were repeated three times using different cDNAs from three transfection experiments. (B) The DNMT3A and DNMT3B mRNA levels in the same cDNA preparation were quantified by using LightCycler real-time PCR. The DNMT3A (left) and DNMT3B (right) levels were expressed relative to GAPDH mRNA levels in the same samples. Results are expressed as the means±S.D. for three experiments. *P<0.05 with the paired t test. (C) Total protein from HEK-293T cells transfected with pCMV-Sp1, pCMV4-Sp3/flu or pRc/CMV was used for Western-blot analyses with anti-Sp1 and anti-Sp3 antibodies respectively. Open arrows indicate the endogenous Sp1 or Sp3 proteins in each Figure. Closed arrows indicate exogenous Sp1 or Sp3 proteins in each Figure.
Figure 3
Figure 3. Identification of the Sp1-binding sites responsible for minimal promoter activities of DNMT3A and DNMT3B in HEK-293T cells
Left panels: schematic structure of the minimal promoter of DNMT3A 3rd promoter pGL3A-P3 (−334/+376) (A), DNMT3B 1st promoter pGL3B-P1 (−102/+309) (B), DNMT3B 2nd promoter pGL3B-P2 (−469/+260) (C) and their deletion constructs are illustrated. The Sp1-binding sites are presented as open boxes and positions are shown relative to the TSP (+1) of each exon. Right panels: the plasmids containing minimal promoter of DNMT3A 3rd promoter, DNMT3B 1st and 2nd promoters and their deletion mutants were transfected into HEK-293T cells. Luciferase activity was expressed relative to the activity of empty reporter vector pGL3-basic. Results are expressed as the means±S.D. for three independent experiments.
Figure 4
Figure 4. Effect of site-specific mutation of Sp1-binding sites on the DNMT3A and DNMT3B promoters
Left panels: schematic representation of site-specific mutation of Sp1-binding sites on minimal promoters of DNMT3A 3rd promoter (A), DNMT3B 1st promoter (B) and DNMT3B 2nd promoter (C). The wild-type and mutant-type Sp1-binding sites are shown as open and filled boxes respectively. Right panels: luciferase activities (%) of the mutant reporter vectors were expressed relative to the activities of their wild-type counterparts. Results are expressed as the means±S.D. for three independent experiments.
Figure 5
Figure 5. Sp1 and Sp3 bind to the 2nd promoter of DNMT3B
The labelled synthetic oligonucleotide spanning nucleotides −119 to −73 relative to the TSP of the DNMT3B 2nd promoter (3B-P2-W1) was incubated with nuclear extract of the HEK-293T cells (lane 2) or in the presence of a 100 or 500 molar excess of the unlabelled wild-type probes (lanes 3 and 4) or the mutant-type probes in which the Sp1-binding site at −100/−92 (5′-GAGGCGGGG-3′) was mutated to 5′-ATATAGGGG-3′ (lanes 5 and 6). For the gel supershift assay, Sp1 and/or Sp3 antibodies were added to the reaction mixture (lanes 7–9). Preimmune serum was used as a control (lane 10). The supershifted bands (S. S.) are indicated.
Figure 6
Figure 6. Down-regulation of DNMT3A and DNMT3B mRNAs with Sp3 antisense oligonucleotide
(A) HEK-293T cells were incubated with either Sp3 antisense oligonucleotide (Sp3-AS) or mismatch oligonucleotide (Sp3-M). Total proteins from both groups were subjected to Western-blot analyses with anti-Sp1 and -Sp3 antibodies. (B) DNMT3A and DNMT3B mRNA levels from antisense and mismatch oligonucleotide-treated cells were quantified with Light-Cycler real-time PCR, then normalized to the GAPDH mRNA levels and expressed relative to the levels in mismatch oligonucleotide-treated cells. *P<0.05 by the t test. Results are expressed as the means±S.D. for three experiments.

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