Table 1.
Primers for qRT-PCR.
Table 2.
Primers for construction of reporter constructs.
Figure 1.
CHD5 expression is down-regulated in leukemia cell lines and samples.
CHD5 expression was determined for K-562, KG-1a, HL-60 and Jurkat cell lines by qRT-PCR (top) and western blotting (bottom) (A). Representative results of CHD5 mRNA (top) and protein (bottom) expression from ALL, AML and CML patients are presented (B). The distribution of CHD5 mRNA (C) and protein level (D) in ALL, AML and CML samples and NMCs (included for comparison) were calculated. For all experiments, β-actin was detected as an internal control. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).
Figure 2.
Mapping regulatory elements within the CHD5 promoter.
The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).
Figure 3.
The location of a CpG island (A) and 39 CpG sites analyzed by BGS (B) in the CHD5 promoter.
CpG sites are indicated with pink vertical bars. The positions analyzed by BGS are indicated with black vertical bars. The TSS is indicated with an arrow.
Figure 4.
The methylation status of the –560 to –240 region of the CHD5 promoter.
The CpG island, located at –560 to –240, was analyzed by BGS. Methylation data from BGS were analyzed by BiQ Analyzer software to generate the lollipop diagram and to calculate the efficiency of bisulfite conversion. Analysis of non-CpG cytosines indicated the efficiency of bisulfite conversion at ∼99%. The lollipop diagram presented the percentage of methylation of each CpG. The overall methylation percentage indicates the total proportion of methylated CpGs in this region taking into account all sequenced alleles.
Figure 5.
The methylation status of CHD5 promoter and corresponding CHD5 expression in leukemia cell lines treated with DAC.
K-562, Jurkat, HL-60 and KG-1a cells were treated with DNA methyltransferase inhibitor DAC at the indicated concentrations. CHD5 expression (A) was analyzed by qPCR (top) and western blotting (bottom). Data are shown as mean ± SD. The methylation status (B) of the –560 to –240 region of the CHD5 promoter was analyzed by BGS.
Figure 6.
Relationship between CHD5 expression level and CHD5 promoter methylation status.
The CpG island located at –560 to –240 was analyzed by BGS. The distribution of overall methylation percentage of CHD5 promoter in ALL, AML and CML and NMCs was calculated (A). The correlation between the methylation level of 30th and 31st CpG sites and CHD5 expression level was analyzed by Spearman’s correlation (B).
Figure 7.
Repression of CHD5 promoter activity by DNA methylation.
A set of reporter constructs of methylated or mock-methylated fragments of the –560 to –240 region were measured for luciferase activity in K-562 cells. The relative Luc activities of methylated and mock CHD5 promoters were normalized to Renilla luciferase activity. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).
Figure 8.
Identification of the AP2 binding site in the CHD5 promoter regulatory element.
ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).