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Since both ITGB4 and ITGA6 responded to DAC treatment, at least in some cell lines, their expression may therefore be regulated by promoter methylation. To determine if the CpG islands located at the ITGB4 and ITGA6 promoters are differentially methylated in the leukaemic cell lines, which express these genes at different levels (Table 5.1), DNA methylation was analysed in K562, KG-1a and Kasumi-1 cells using bisulphite sequencing.

Genomic DNA was isolated from K562, KG-1a and Kasumi-1 cells and was subjected to bisulphite conversion, as described in Chapter 2, Section 2.6. PCR primers were designed to amplify the region -516 bp to +276 bp of ITGB4, encompassing 85 CpG sites. PCR products were cloned into the pGEM-T Easy vector and single clones were selected for sequencing. Interestingly, the ITGB4 promoter was found to be differentially methylated in the cell lines examined. The promoter was highly methylated in KG-1a cells, but was relatively unmethylated in K562 and Kasumi-1 cells (Figure 5.4). The DNA methylation pattern in KG-1a cells was striking, while there was some methylation upstream of the transcription start site (including through the RUNX1 responsive region), the region downstream of the transcription start site was completely methylated (Figure 5.4). Taken together, these data suggest that in both K562 and Kasumi-1 cells the ITGB4 promoter is likely to be relatively accessible, whereas the ITGB4 promoter in KG-1a cells is likely to be less accessible due to higher levels of DNA methylation. DNA methylation is generally associated with gene silencing, and therefore the methylation patterns observed at the promoter in KG-1a and Kasumi-1 cells reflects the expression of ITGB4 in these cells (ie. expression in Kasumi-1 but not KG-1a), whereas in K562 cells the ITGB4 promoter is largely unmethylated which does not explain the low expression of ITGB4 in these cells (Table 5.1).

To determine how the methylation pattern of the ITGB4 promoter in leukaemic cell lines compares to human samples, DNA methylation of the ITGB4 promoter was analysed in whole blood samples from individuals with (LK7770) and without leukaemia (LK004 and LK124). Interestingly, the ITGB4 promoter was relatively unmethylated in all samples (Figure 5.5) and most similar to the pattern seen in Kasumi-1 cells. While the leukaemic sample, which is positive for the t(8;21) chromosomal translocation, showed

143 a similar methylation pattern to the t(8;21)-positive Kasumi-1 cell lines, with most methylation located at the 3’ end (Figure 5.5), this pattern was also seen in the non- leukaemic cells. These data therefore suggest that the ITGB4 promoter is normally unmethylated in human blood cells, and the presence of the RUNX1-ETO fusion protein does not appear to have an effect on the DNA methylation pattern at the ITGB4 promoter. DNA methylation of the ITGB4 promoter was also analysed in treated and untreated cell lines to determine if DAC caused demethylation at this region. K562, KG-1a and Kasumi- 1 cells were treated with DAC for 72 hours. Genomic DNA was isolated and subjected to bisulphite conversion, as described in Chapter 2, Section 2.6. Due to difficulty in amplifying the region -516 bp to +276 bp from DAC treated cells, a different set of PCR primers were used to amplify a smaller region of the ITGB4 CpG island, located -390 bp to +276 bp of ITGB4, encompassing 72 CpG sites. The PCR products were cloned into the pGEM-T Easy vector and single clones were sequenced. Treatment of cells with DAC had no effect in K562 cells, which is unsurprising because the ITGB4 CpG island is unmethylated in these cells (Figure 5.6). Kasumi-1 cells displayed demethylation at the 3’ end, where most of the methylation is observed (Figure 5.6). Surprisingly, DAC treatment had no effect on DNA methylation of the ITGB4 promoter in KG-1a cells, although this region is heavily methylated in these cells (Figure 5.6).

144 Figure 5.4 – DNA methylation patterns at the ITGB4 CpG island in leukaemic cell lines. CpG sites located in a region of -516 bp to +276 bp of ITGB4 within the ITGB4 promoter CpG island were analysed for methylation in K562, KG-1a and Kasumi-1 cell lines using bisulphite sequencing. Sequencing was analysed using BiQ Analyzer software and bubble maps were generated using CpG Bubble Chart Generator, Version 20061209 (created by Mark A. Miranda). Each line represents a single clone and circles represent CpG sites. White circles represent unmethylated CpG sites whereas black circles represent methylated sites and crosses are CpG sites with a mutation. Schematic representation of the ITGB4 promoter is shown above and the scale represents base pairs relative to the transcription start site (indicated by arrow).

145 Figure 5.5 – DNA methylation patterns at the ITGB4 CpG island in t(8;21)-positive leukaemia and non-leukaemia samples. CpG sites located in a region of -516 bp to +276 bp of ITGB4 within the ITGB4 promoter CpG island were analysed for methylation in non-leukaemia (LK004 and LK124) and t(8;21)-positive leukaemia individual samples (LK7770) using bisulphite sequencing. Sequencing was analysed using BiQ Analyzer software and bubble maps were generated using CpG Bubble Chart Generator, Version 20061209 (created by Mark A. Miranda). Each line represents a single clone and circles represent CpG sites. White circles represent unmethylated CpG sites whereas black circles represent methylated sites and crosses are CpG sites with a mutation. Schematic representation of the ITGB4 promoter is shown above and the scale represents base pairs relative to the transcription start site (indicated by arrow).

146 Figure 5.6 - DNA methylation patterns at the ITGB4 CpG island in untreated and DAC treated leukaemic cell lines. CpG sites located in a region of -390 bp to +276 bp of ITGB4 within the ITGB4 promoter CpG island were analysed for methylation in untreated and DAC treated K562, KG-1a and Kasumi-1 cell lines using bisulphite sequencing. Sequencing was analysed using BiQ Analyzer software and bubble maps were generated using CpG Bubble Chart Generator, Version 20061209 (created by Mark A. Miranda). Each line represents a single clone and circles represent CpG sites. White circles represent unmethylated CpG sites whereas black circles represent methylated sites and crosses are CpG sites with a mutation. Schematic representation of the ITGB4 promoter is shown above and the scale represents base pairs relative to the transcription start site (indicated by arrow).

147 To determine if the ITGA6 promoter CpG island, similar to the ITGB4 promoter CpG island, is differentially methylated in the leukaemic cell lines and if DAC treatment causes demethylation of the CpG island, bisulphite sequencing was similarly used to analyse CpG methylation at the promoter. K562, KG-1a and Kasumi-1 cells were treated with DAC for 72 hours. Genomic DNA was isolated from treated and untreated cells, and subjected to bisulphite conversion, as described in Chapter 2, Section 2.6. PCR primers were designed to amplify the region surrounding the ITGA6 transcription start site in two sections located -329 bp to +240 bp (Fragment A) and +216 bp to +821 bp (Fragment B). PCR products were cloned in the pGEM-T easy vector and single clones were selected for sequencing.

Fragment A, which incorporates the transcription start site of ITGA6, was largely unmethylated in all leukaemic cell lines analysed (Figure 5.7). Not surprisingly then, DAC treatment did not result in any significant decrease in methylation at this region (Figure 5.7). Similarly, Fragment B, which incorporates the gene body adjacent to the transcription start site, including the translation start site, was largely unmethylated, although there was some methylation towards the 3’ end of the region in K562 cells (Figure 5.8). Similar to Fragment A, treatment with DAC had little effect on methylation of Fragment B due to the existing low levels of methylation (Figure 5.8).

Taken together, these data suggest that the ITGA6 promoter is likely to be relatively accessible in K562, KG-1a and Kasumi-1 cell lines due to low levels of DNA methylation. Unmethylated promoters are typically associated with actively transcribed genes, and therefore the methylation patterns observed at the ITGA6 promoter in KG-1a and Kasumi-1 cells reflects expression of ITGA6 in these cells (ie. expressed in both cell lines), whereas in K562 cells, the ITGA6 promoter is largely unmethylated which does not explain the low expression of ITGA6 in these cells (Table 5.1).

148 Figure 5.7 – DNA methylation patterns of Fragment A at the ITGA6 CpG island in untreated and DAC treated leukaemic cell lines. CpG sites located in a region of -329 bp to +240 bp of ITGA6 within the ITGA6 promoter CpG island were analysed for methylation in untreated and DAC treated K562, KG-1a and Kasumi-1 cell lines using bisulphite sequencing. Sequencing was analysed using BiQ Analyzer software and bubble maps were generated using CpG Bubble Chart Generator, Version 20061209 (created by Mark A. Miranda). Each line represents a single clone and circles represent CpG sites. White circles represent unmethylated CpG sites whereas black circles represent methylated sites and crosses are CpG sites with a mutation. Schematic representation of the ITGA6 promoter is shown above and the scale represents base pairs relative to the transcription start site (indicated by arrow).

149 Figure 5.8 – DNA methylation patterns of Fragment B at the ITGA6 CpG island in untreated and DAC treated leukaemic cell lines. CpG sites located in a region of +216 bp to +821 bp of ITGA6 within the ITGA6 promoter CpG island were analysed for methylation in untreated and DAC treated K562, KG-1a and Kasumi-1 cell lines using bisulphite sequencing. Sequencing was analysed using BiQ Analyzer software and bubble maps were generated using CpG Bubble Chart Generator, Version 20061209 (created by Mark A. Miranda). Each line represents a single clone and circles represent CpG sites. White circles represent unmethylated CpG sites whereas black circles represent methylated sites and crosses are CpG sites with a mutation. Schematic representation of the ITGA6 promoter is shown above and the scale represents base pairs relative to the transcription start site (indicated by black arrow). Translation start site is indicated by the green arrow.

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