Figure 6
Figure 6. Results of ChIP-chip and their validation by quantitative PCR. (A) ChIP-chip analysis using the I12 anti-HOXB4 monoclonal antibody revealed that there was increased binding to the promoter regions of the B220, CD34, Sox4, Laptm4b, Gp49a, and HMBS genes by the anti-HOXB4 IP, compared with input. In contrast, the control IP obtained using antirat IgG2a antibody beads alone showed no comparable increased binding. By microarray analysis, the Khdrbs1 gene was not shown to be differentially expressed in KLS-EML-HOXB4 cells, and no binding of HOXB4 to its promoter region was detected by ChIP; it is shown as a negative control. *The promoter region for the erythroid-specific isoform of HMBS, which is located within the sequence region of the first intron of the housekeeping isoform of HMBS. Interestingly, there is a greater degree of increased binding of HOXB4 to the promoter region for the erythroid-specific isoform of HMBS than to the promoter region for the housekeeping isoform of HMBS. Arrows represent transcription start sites of the genes. (B) Primers were designed corresponding to the promoter regions of the negative control Khdrbs1 gene and of 17 genes that were observed by ChIP-chip to be enriched at least 2-fold in the IP obtained with the I12 anti-HOXB4 monoclonal antibody. Validation of the enrichment was carried out by quantitative real-time PCR. The PCR results confirmed that 15 of the 17 selected promoter regions were indeed enriched in these IPs compared with the input. Results using the IPs obtained using the control beads conjugated to antirat IgG2a antibody are shown for comparison. Values are mean plus or minus SD of triplicate PCR analyses of ChIP DNA from the 3 different single-cell clones of KLS-EML-HOXB4 cells. (C) Top-scoring DNA-binding motif identified, using the software program Weeder (Version 1.3), in an unbiased analysis of the top 1000 promoter sequences bound by HOXB4 in the ChIP-chip experiments. The known HOX protein binding consensus sequence, TAAT, is found prominently in this motif.

Results of ChIP-chip and their validation by quantitative PCR. (A) ChIP-chip analysis using the I12 anti-HOXB4 monoclonal antibody revealed that there was increased binding to the promoter regions of the B220, CD34, Sox4, Laptm4b, Gp49a, and HMBS genes by the anti-HOXB4 IP, compared with input. In contrast, the control IP obtained using antirat IgG2a antibody beads alone showed no comparable increased binding. By microarray analysis, the Khdrbs1 gene was not shown to be differentially expressed in KLS-EML-HOXB4 cells, and no binding of HOXB4 to its promoter region was detected by ChIP; it is shown as a negative control. *The promoter region for the erythroid-specific isoform of HMBS, which is located within the sequence region of the first intron of the housekeeping isoform of HMBS. Interestingly, there is a greater degree of increased binding of HOXB4 to the promoter region for the erythroid-specific isoform of HMBS than to the promoter region for the housekeeping isoform of HMBS. Arrows represent transcription start sites of the genes. (B) Primers were designed corresponding to the promoter regions of the negative control Khdrbs1 gene and of 17 genes that were observed by ChIP-chip to be enriched at least 2-fold in the IP obtained with the I12 anti-HOXB4 monoclonal antibody. Validation of the enrichment was carried out by quantitative real-time PCR. The PCR results confirmed that 15 of the 17 selected promoter regions were indeed enriched in these IPs compared with the input. Results using the IPs obtained using the control beads conjugated to antirat IgG2a antibody are shown for comparison. Values are mean plus or minus SD of triplicate PCR analyses of ChIP DNA from the 3 different single-cell clones of KLS-EML-HOXB4 cells. (C) Top-scoring DNA-binding motif identified, using the software program Weeder (Version 1.3), in an unbiased analysis of the top 1000 promoter sequences bound by HOXB4 in the ChIP-chip experiments. The known HOX protein binding consensus sequence, TAAT, is found prominently in this motif.

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