Figure 3
Figure 3. Genetic and epigenetic abnormalities of the TCF8 gene in ATLL cells. (A) Bisulfite genomic sequencing of the TCF8 promoter region in 3 ATLL cell lines: ED, KOB, and KK1. PCR products amplified from bisulfite-treated DNA were subcloned, and 8 clones in each cell line were sequenced. ○ indicate unmethylated CpGs (Thy), and • indicate methylated CpGs (Cyt). The sequenced region contains 50 CpGs in 632 bp, just upstream from exon 1. Pro indicates a region of the TCF8 promoter for chromatin immunoprecipitation. (B) Specific DNA binding of acetylated histone H3 or H4 to the TCF8 promoter region detected by chromatin immunoprecipitation (ChIP). Two genomic DNA fragments containing every possible DNA-binding site, TCF8 promoter, or β-actin promoter were amplified from the genomic DNA of fixed ATLL-related cell lines (MT2, HUT102, ED, and KK1) after immunoprecipitation with normal rabbit serum (NRS) or with antiacetylated histone H3 or H4 antibodies (αH3 or αH4). Quantitative PCR data calibrated to the TCF8 promoter/β-actin ratio are shown in the NRS as a relative expression rate of 1. Data are the means plus or minus standard deviation of 2−ΔΔCt in a duplicate assay. Cell lines were cultured in RPMI1640 medium containing 10% FCS with (+ TSA) or without (control) 1.2 μM TSA. (C) Genomic structure of the TCF8 gene with a missense mutation and single nucleotide polymorphisms. Locations of the mutations and the single nucleotide polymorphisms relative to the exons encoding the functional domains are shown. TCF8 encodes a homeodomain (HD) flanked by 2 zinc-finger clusters (z1 to z4 and z5 to z7) (NZF indicates N-terminal zinc finger repeats, CZF; C-terminal zinc finger repeats). The N-terminal transcriptional regulatory domain (N-reg) could bind to p300/CBP and the C-terminal transcriptional regulator domain (C-reg) is the region where acidic amino acids are clustered just after the last zinc-finger domain. Values represent the number of mutated cases per total number of tested cases. SNP indicates single nucleotide polymorphism. White boxes represent noncoding regions in exons 1 and 9.

Genetic and epigenetic abnormalities of the TCF8 gene in ATLL cells. (A) Bisulfite genomic sequencing of the TCF8 promoter region in 3 ATLL cell lines: ED, KOB, and KK1. PCR products amplified from bisulfite-treated DNA were subcloned, and 8 clones in each cell line were sequenced. ○ indicate unmethylated CpGs (Thy), and • indicate methylated CpGs (Cyt). The sequenced region contains 50 CpGs in 632 bp, just upstream from exon 1. Pro indicates a region of the TCF8 promoter for chromatin immunoprecipitation. (B) Specific DNA binding of acetylated histone H3 or H4 to the TCF8 promoter region detected by chromatin immunoprecipitation (ChIP). Two genomic DNA fragments containing every possible DNA-binding site, TCF8 promoter, or β-actin promoter were amplified from the genomic DNA of fixed ATLL-related cell lines (MT2, HUT102, ED, and KK1) after immunoprecipitation with normal rabbit serum (NRS) or with antiacetylated histone H3 or H4 antibodies (αH3 or αH4). Quantitative PCR data calibrated to the TCF8 promoter/β-actin ratio are shown in the NRS as a relative expression rate of 1. Data are the means plus or minus standard deviation of 2−ΔΔCt in a duplicate assay. Cell lines were cultured in RPMI1640 medium containing 10% FCS with (+ TSA) or without (control) 1.2 μM TSA. (C) Genomic structure of the TCF8 gene with a missense mutation and single nucleotide polymorphisms. Locations of the mutations and the single nucleotide polymorphisms relative to the exons encoding the functional domains are shown. TCF8 encodes a homeodomain (HD) flanked by 2 zinc-finger clusters (z1 to z4 and z5 to z7) (NZF indicates N-terminal zinc finger repeats, CZF; C-terminal zinc finger repeats). The N-terminal transcriptional regulatory domain (N-reg) could bind to p300/CBP and the C-terminal transcriptional regulator domain (C-reg) is the region where acidic amino acids are clustered just after the last zinc-finger domain. Values represent the number of mutated cases per total number of tested cases. SNP indicates single nucleotide polymorphism. White boxes represent noncoding regions in exons 1 and 9.

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