The cloning and characterization of the histone demethylase Dmel\JHDM3A during Drosophila melanogaster development

Madhusmita Datta

doi:10.17918/etd-2782

Core histone proteins of the nucleosome undergo covalent modifications such as acetylation, methylation, ubiquitination and phosphorylation at their N terminal tails. These modifications regulate chromatin structure and gene transcription. Until the recent discovery of histone demethylases (HDMs), histone methylation was considered to be a permanent modification. Three families of HDMs have been identified that antagonize histone methylation through distinctive mechanisms [1, 2] [3, 4]. The wide ranging impact of histone methylation on biological processes such as formation of heterochromatin, homeotic gene silencing and transcriptional regulation [5] underscores the importance of the demethylation process. In addition, a number of newly discovered HDMs appear to play an important role in tumorigenesis, therefore HDMs represent a promising target for cancer therapy[6-9]. We are therefore interested in examining the role of the Drosophila homolog of human JHDM3A/JMJD2A - the first trimethyl specific HDM discovered, during Drosophila development. Here we report the first cloning and functional characterization Dmel\JHDM3A. We have used a P-element suppressor fly line to examine the affect of disruption of the gene Dmel\JHDM3A on various aspects of Drosophila development. Interestingly, we observe that mutant flies display a twitching phenotype reminiscent of muscle and/or nervous system disruption [10, 11]. We have successfully generated two revertant fly lines through genetic crosses that have a precise excision of the P-element in the Dmel\JHDM3A gene. Importantly, these fly lines show no evidence of twitching, indicating that this phenotype is caused by the disruption of the Dmel\JHDM3A gene. However, embryonic antibody staining with neuronal antibodies revealed no observable defects in nervous system during early development and climbing assay indicated no obvious defects in the climbing ability during adult stages, suggesting that the twitching behavior could be due either to muscular irregularities or due to subtle defects in the neuronal system that were not captured by the assays conducted. We observe through RT-PCR experiments that HDMs from two different families are expressed during various stages of development, indicative of the requirement and therefore the role of the various HDMs during Drosophila development. Additionally, longevity assay revealed that mutant male flies have a significantly shorter mean lifespan compared to wild-type flies. Furthermore, the preference for oviposition site is altered in the mutant flies such that the mutant females lay eggs away from food, which may not be in favor of the survival of the emerging larvae. This may be due to olfactory defects suggestive of an underlying neurological cause [12]. Dmel\JHDM3A did not show positive chromatin modifying effects as revealed through position effect variegation experiments. This does not however rule out the possibility that Dmel\JHDM3A contributes to chromatin modification in a subtle way that was not detected through PEV experiment. Taken together the results suggest that Dmel\JHDM3A plays specific roles in distinct processes during Drosophila development.

The cloning and characterization of the histone demethylase Dmel\JHDM3A during Drosophila melanogaster development

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The cloning and characterization of the histone demethylase Dmel\JHDM3A during Drosophila melanogaster development

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