Gene expression changes of dTip60^E431Q misregulated target genes in the different transgenic lines.

a<p>Quantitative RT-PCR analysis was performed for the indicated target genes.</p>b<p>Staged second instar larvae ubiquitously expressing the indicated transgene(s) were used for cDNA preparation. Quantitative RT-PCR reactions were carried out in triplicate and the relative fold change was calculated using the 2−ΔΔCT method using RP49 as control.</p>§<p>Genes that were differentially regulated between flies expressing the Tip60 HAT mutant dTip60^E431Q alone and in conjunction with APP.</p> <p>Gene that were differentially regulated between flies overexpressing dTip60^WT alone or together with APP.</p

Viability analysis indicates genetic interaction between Tip60 and APP in <i>Drosophila</i>.

<p>The indicated transgene was expressed ubiquitously in the fly using 337-Gal4 driver or pan-neuronally using 179 y-Gal4 driver. The number of F1 progeny that eclosed were counted daily. The percentage of eclosed flies was calculated relative to the wild type control (<i>w¹¹¹⁸</i>). All crosses were carried out in triplicate at 25°C. Overexpression of APP drastically reduced viability to <10% while no effect was observed due to expression of truncated version of APP lacking its C-terminal domain. Overexpression of varying levels of wild type dTip60 (dTip60^WT) also reduced viability in a dose independent manner. However, co-expression of dTip60^WT with APP partially rescued the lethal effects induced by APP expression in a dose dependent manner with the maximum effect observed with high levels of dTip60^WT. In the presence of APP lacking the C-terminus, overexpression of dTip60^WT had similar effects seen in flies that overexpressed dTip60^WT alone.</p

Transgenic fly lines used for this study.

a<p>The Tip60 P-element insertion is located on chromosome 3 and the APP P-element insertion is located on chromosome 2.</p>b<p>Indicates where the transgenic fly lines were generated.</p

Generation and characterization of dTip60^E431Q containing APP or APP-dCT double transgenic flies.

<p>The dominant negative HAT defective lines dTip60^E431Q A or dTip60^E431Q B (Lorbeck <i>et al.</i>, 2011) were introduced into an APP or APP dCT background using standard genetic techniques. (<b>A</b>) Histogram depicting qPCR analysis of exogenous levels of dTip60^E431Q in staged F1 second instar larval progeny resulting from a cross between the ubiquitous driver 337 and either dTip60^E431Q (lines A and B), APP; dTip60^E431Q (lines A and B) or APP dCT; dTip60^E431Q (lines A and B). 337-Gal4 crossed to <i>w¹¹¹⁸</i> served as a control. Quantification of the exogenously expressed dTip60^E431Q mRNA levels relative to endogenously expressed dTip60 mRNA was done using the comparative CT method with RP49 as internal control as described in (Lorbeck <i>et al</i>, 2011). Asterisks (*) indicate significant fold change between the lines A and B for each genotype with values of p<0.05; n = 3. Error bars represent standard error of the mean. (<b>B</b>) Semiquantitative RT-PCR analysis of APP or APP dCT expression in the different transgenic lines to confirm APP transgene presence. cDNA was prepared as before from staged second instar larvae ubiquitously expressing dTip60^E431Q with APP or APP dCT (lines A or B in each case) and PCR amplified using primers that flank a 100 bp region in the N-terminal portion of APP. PCR products were visualized using 2% agarose gel containing ethidium bromide. Staged second instar larvae ubiquitously expressing APP or APP dCT were used as controls.</p

Generation and characterization of dTip60^WT containing APP or APP-dCT double transgenic flies.

<p>Flies expressing varying levels of wild type dTip60 (low, medium and high) were generated and then each introduced into APP or APP dCT background using standard genetic techniques. (<b>A</b>) The amount of wild type dTip60 that is exogenously induced relative to endogenous dTip60 was quantified by RT-PCR analysis of staged F1 second instar larvae resulting from the a cross between the ubiquitous driver 337 and either dTip60^WT (lines A, B and C), APP; dTip60^WT (lines A, B and C) or APP dCT; dTip60^WT (lines A, B and C). 337-Gal4 crossed to <i>w¹¹¹⁸</i> was used as control. The relative fold change in mRNA expression levels between exogenous and endogenous dTip60 was measured as described before using the comparative CT method with RP49 as the internal control, and these results are summarized in the histogram. The amount of exogenously induced wild type dTip60 levels is significantly different between lines A, B and C in each case with values of p<0.05; n = 3. Error bars represent standard error of the mean. (<b>B</b>) Semi-quantitative RT-PCR analysis of APP or APP dCT expression in the different dTip60^WT containing transgenic lines to confirm APP transgene presence. cDNA was prepared as before from staged second instar larvae ubiquitously expressing dTip60^WT with APP or APP dCT (lines A, B or C in each case) and PCR amplified using primers that flank a 100 bp region in the N-terminal portion of APP. PCR products were visualized using 2% agarose gel containing ethidium bromide. Staged second instar larvae ubiquitously expressing APP or APP dCT were used as controls.</p

Quantitative RT-PCR validation of selected apoptosis related genes identified by microarray analysis.

<p>(<b>A</b>) Histogram showing relative fold change in expression level of apoptosis related target genes in flies expressing dTip60^E431Q A. Staged second instar larvae were used for cDNA preparation. RT-PCR reactions were carried out in triplicate and the fold change was calculated using the 2−ΔΔCT method using RP49 as control. (<b>B</b>) List of selected apoptosis related target genes identified by microarray analysis and validated in the dTip60^E431Q A line using quantitative RT-PCR.</p

Apoptosis pathways significantly misregulated in response to dTip60 HAT loss.

<p>Apoptosis pathways significantly misregulated in response to dTip60 HAT loss.</p