Mutant analysis identifies E93 regulated genes in salivary gland cell death

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To identify the genes with differential expression that is most likely associated with the autophagic cell death process, we carried out E93 mutant analyses.   E93 expression appears to specifically foreshadow steroid-induced cell death [6] and E93 mutant salivary glands display morphological features indicative of a block in the early stages of autophagic cell death [3].  Further, the ecdysone-induced genes, BR-C, E74, and E75, and the cell death genes, rpr, hid, crq, and dronc, are all transcribed at reduced levels in E93 mutant salivary glands [6].  E93 encodes a novel nuclear protein that binds to multiple sites on larval salivary gland polytene chromosomes [6].  The map position of crq correlates with an E93 binding site [6] and it may thus be regulated directly by E93.  To identify other genes that may be regulated transcriptionally by E93 in salivary gland death, we screened all differentially expressed genes for those with a map position corresponding to E93 binding sites.  We identified 43 upregulated genes and 41 downregulated genes corresponding to 39 of the 65 known E93 binding sites (data not shown).  To test further whether these genes may be regulated directly by E93, we analyzed their transcription profiles in E93 mutant salivary glands.  Since previous studies indicated a role for E93 as a positive regulator of cell death gene expression, we tested genes upregulated significantly at 23 hrs APF.  Of 18 confirmed upregulated genes tested, all but one (Sox14) exhibited a reduction in the fold-difference in expression in the E93 mutant background compared to control genes (Figure 1).   These results indicate that these 17 genes are regulated by E93, indirectly or directly, and that their expression is thus likely associated specifically with autophagic cell death.