ENCUESTA A USUARIOS DE VÍDEO CABLE PREGUNTA Nº 1

In a first instance, the toxin okadaic acid, which strongly inhibits serine/threonine phosphatases, was used in order to increase the abundance of phosphorylated histone marks by preventing their removal. Undifferentiated HPC-7 cells were treated with a range of okadaic acid concentrations, and a time-course experiment was performed, in order to determine the effect of this agent on global histone modifications. Cells were treated with 1, 5 or 10nM okadaic acid, histones were extracted and histone modification abundance quantified by Western blotting using a range of appropriate antibodies including H3K9acS10p (Ab12181), H3S10p (Ab14955), H3T3p (Ab17352). We also analysed the non- phospho-isoform H4K16ac (Lab 252) as a control to examine whether okadaic acid only has impacts on histone phosphorylation (Figure 6.3a). The bottom panel shows after Coomassie staining that histones were equally loaded (“Total histone”), though Western analysis with a Histone H3 C-terminal antibody (AbCam) was used to normalise for protein loading. All marks examined show an increased abundance following the addition of a gradient of okadaic acid (Figure 6.3b). The highest increase is observed for the dual mark H3K9acS10p, which shows a #2.5-fold rise, whilst H3S10p is only slightly increased (It appears to be reduced due to unequal histone loading). In addition, another phosphorylated mark, H3T3p, shows a #2-fold increase after treatment with 10nM okadaic acid, confirming that the agent is not selective for serine-specific phosphatases. Perhaps not surprisingly, H4K16ac abundance, which would not be expected to increase as a direct effect of okadaic acid, also shows a small

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! Figure 6.3: Quantification of histone modifications after exposure to gradient of concentration of okadaic acid in undifferentiated HPC-7 cells Undifferentiated HPC-7 cells were treated with 1, 5 or 10nM of okadaic Acid for 24 hours. Histones were extracted and specific marks were examined by (a) Western blotting using appropriate antibodies (H3K9acS10p, H3T3p, H4K16ac and H3S10p). The gel is shown to be equally loaded (SDS gel). (b) Quantification is presented on a graph, normalised to the control, set at 1.

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increase, suggesting that either “histone cross-talk” effects, or secondary, indirect gene effects take place after treatment.

Subsequently, a time-course experiment with 10nM okadaic Acid treatment of undifferentiated HPC-7 cells was performed in order to identify the ideal window of treatment with this toxin. Undifferentiated HPC-7 cells were treated with 10nM okadaic acid for 1, 2, 4, 8, 12, 18 and 24 hours. Histone were extracted as previously described and their abundance was quantified by Western blotting using H3K9acS10p or H4K16ac antibodies (Figure 6.4), using detection by the H3 C-terminal antibody (AbCam) to normalise for protein loading. As shown before, H4K16ac shows a small change (10% increase) during the time- course. By this assessment, global H3K9acS10p modification seems to be the highest between four and eight hours treatment.

In undifferentiated HPC-7 cells, active Hoxa genes were associated with H3S10p enrichment over the promoter of genes, whilst a reduction of Hoxa gene expression in megakaryocytes was associated with the loss of this mark. We hypothesised that the increase in H3S10P abundance due to okadaic acid treatment would lead to an increase of MLL target gene expression in both undifferentiated HPC-7 cells and megakaryocytes (Figure 6.5). We therefore examined the transcript abundance of specific Hoxa genes following treatment of undifferentiated HPC-7 cells and megakaryocytes with 1, 5 or 10nM okadaic acid for 8 (data not shown) and 24 hours (Figure 6.6). After eight hours of treatment, slight changes of the level of MLL target gene expression in undifferentiated HPC-7 cells were observed, with only small changes at Hoxa4 and Gapdh (1.2 and 1.5-fold changes respectively). No changes were observed with Hoxa2, Hoxa3 and Hoxa5.

Changes in the level of gene expression following 24 hours of 10nM okadaic acid treatment were then examined in undifferentiated HPC-7 cells and megakaryocytes (Figure 6.6). The

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! Figure 6.4: Effect of okadaic acid on histone modification abundance in undifferentiated HPC-7 cells

Undifferentiated HPC-7 cells were treated with 10nM okadaic acid for 1, 2, 4, 8, 12, 18 and 24 hours. Histones were extracted and specific marks were examined by Western blotting using appropriate antibodies (H3K9acS10p, and H4K16ac). Mark abundance is normalised to untreated control cells.

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! Figure 6.5: Model of changes possible after treatment with okadaic acid

In undifferentiated HPC-7 cells, active Hoxa genes are associated with a H3S10p enrichment over the promoter of genes, whilst a reduction of Hoxa gene expression in megakaryocytes is associated with the loss of this mark. In this model, it is suggested that the prevention of phosphate removal at H3S10 by the phosphatase inhibitor, okadaic acid, would lead to an increase of gene expression in both undifferentiated HPC-7 cells and megakaryocytes.

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expression of several genes (Hoxa2, Hoxa3, Hoxa4, Hoxa5 and Gapdh) was examined in response to this treatment, where gene expression is normalised with the level of expression in control cells (cells treated with DMSO). In undifferentiated cells (Figure 6.6, Upper panel), an increase of gene expression Hoxa4 and Gapdh (1.4 and 3-fold respectively) was observed in correlation with an increased concentration of okadaic acid. No changes were observed for Hoxa2, Hoxa3 and Hoxa5. In megakaryocytes (Figure 6.6, Lower panel), only Hoxa3 showed a 8-fold increase of its level of expression following okadaic acid treatment, possibly reflecting the lower abundance of this transcript in megakaryocytes. No changes were observed for Hoxa2, Hoxa4, Hoxa5 and Gapdh. It should be noted that the addition of okadaic acid was performed after five days of megakaryocyte differentiation, suggesting that the Hoxa genes would be expected to have already lost H3S10 phosphorylation, as the genes are down-regulated.

6.2.2. What are the local changes on Hoxa genes after treatment with okadaic acid?