Figure 4.
Figure 4. EKLF occupies conserved CACC elements in dematin upstream gene regulatory regions and directly activates 2 dematin promoters. (A) Schematic of organization of the dematin gene. The original cDNA starts at exon 3.38 The other putative upstream exons correspond to alternatively spliced transcripts. (B) An RT-PCR analysis of alternative transcript used in E14.5 fetal liver. The arrows represent RT-PCR primers. (C) Schematic of the ECR regions of the dematin genomic sequence. ECR regions are defined as areas of greater than 70% sequence identity of at least 100 bp in length and are numbered in reverse order from intron 2. All putative EKLF and GATA-1 sites within this region are marked, as are the conserved sites between the human, mouse, and rat genomes. A plot of percentage identity between human and mouse sequences is shown, with red regions corresponding to ECRs. The positions of exons 1 and 2 are indicated for reference. (D) ChIP using EKLF antisera or nonimmune sera. Plots show real-time PCR analysis of the precipitated DNA from the corresponding upstream regions. For each region, ChIPed material is expressed relative to input DNA. ChIP was performed on B1.6 cells treated in 4 ways: using EKLF-specific or preimmune sera and after tamoxifen-induced (T) or control (E) induction of EKLF-ER. (E) ChIP using ERα monoclonal antibody or isotype control. Again, ChIP was performed on cells treated in 4 ways: using ERα antibody or mIgG1 and after tamoxifen-induced (T) or control (E) induction of EKLF-ER. (F) Mean relative luciferase levels (as normalized to renilla) ± SEM, n = 3. pGL3-basic (Luc) constructs were transfected into SL2 cells with or without an EKLF expression vector. In each case transfection of the Luc construct alone did not alter luciferase levels (data not shown). EKLF response data are presented normalized to 1 for each construct. The βmaj-Luc contains the β-major globin promoter and is a positive control for EKLF action. DemP3 corresponds to -1 to -1000, and DemP2 to -8267 to -9269 from the transcription start site of the dematin gene.38

EKLF occupies conserved CACC elements in dematin upstream gene regulatory regions and directly activates 2 dematin promoters. (A) Schematic of organization of the dematin gene. The original cDNA starts at exon 3.38  The other putative upstream exons correspond to alternatively spliced transcripts. (B) An RT-PCR analysis of alternative transcript used in E14.5 fetal liver. The arrows represent RT-PCR primers. (C) Schematic of the ECR regions of the dematin genomic sequence. ECR regions are defined as areas of greater than 70% sequence identity of at least 100 bp in length and are numbered in reverse order from intron 2. All putative EKLF and GATA-1 sites within this region are marked, as are the conserved sites between the human, mouse, and rat genomes. A plot of percentage identity between human and mouse sequences is shown, with red regions corresponding to ECRs. The positions of exons 1 and 2 are indicated for reference. (D) ChIP using EKLF antisera or nonimmune sera. Plots show real-time PCR analysis of the precipitated DNA from the corresponding upstream regions. For each region, ChIPed material is expressed relative to input DNA. ChIP was performed on B1.6 cells treated in 4 ways: using EKLF-specific or preimmune sera and after tamoxifen-induced (T) or control (E) induction of EKLF-ER. (E) ChIP using ERα monoclonal antibody or isotype control. Again, ChIP was performed on cells treated in 4 ways: using ERα antibody or mIgG1 and after tamoxifen-induced (T) or control (E) induction of EKLF-ER. (F) Mean relative luciferase levels (as normalized to renilla) ± SEM, n = 3. pGL3-basic (Luc) constructs were transfected into SL2 cells with or without an EKLF expression vector. In each case transfection of the Luc construct alone did not alter luciferase levels (data not shown). EKLF response data are presented normalized to 1 for each construct. The βmaj-Luc contains the β-major globin promoter and is a positive control for EKLF action. DemP3 corresponds to -1 to -1000, and DemP2 to -8267 to -9269 from the transcription start site of the dematin gene.38 

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