A mechanistic understanding of the natural aging process, which is distinct from aging-related disease mechanisms, is essential for developing interventions to extend lifespan or healthspan. Here, we discuss current trends in aging research and address conceptual and experimental challenges in the field. We examine various molecular markers implicated in aging with an emphasis on the role of heterochromatin and epigenetic changes. Studies in model organisms have been advantageous in elucidating conserved genetic and epigenetic mechanisms and assessing interventions that affect aging. We highlight the use of Drosophila, which allows controlled studies for evaluating genetic and environmental contributors to aging conveniently. Finally, we propose the use of novel methodologies and future strategies using Drosophila in aging research.

Background

The Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway has been well-characterized as a crucial signal transduction cascade that regulates vital biological responses including development, immunity and oncogenesis. Additionally to its canonical pathway that uses the phosphorylated form of the STAT transcription factor, recently the non-canonical pathway involving heterochromatin formation by unphosphorylated STAT was recently uncovered. Considering the significant role of the JAK/STAT pathway, we used the simple Drosophila system in which the non-canonical pathway was initially characterized, to compare putative canonical versus non-canonical transcriptional targets across the genome. We analyzed microarray expression patterns of wildtype, Jak gain- and loss-of-function mutants, as well as the Stat loss-of-function mutant during embryogenesis, since the contribution of the canonical signal transduction pathway has been well-characterized in these contexts. Previous studies have also demonstrated that Jak gain-of-function and Stat mutants counter heterochromatin silencing to de-repress target genes by the non-canonical pathway.

Results

Compared to canonical target genomic loci, non-canonical targets were significantly more associated with sites enriched with heterochromatin-related factors (p = 0.004). Furthermore, putative canonical and non-canonical transcriptional targets identified displayed some differences in biological pathways they regulate, as determined by Gene Ontology (GO) enrichment analyses. Canonical targets were enriched mainly with genes relevant to development and immunity, as expected, whereas the non-canonical target gene set mainly showed enrichment of genes for various metabolic responses and stress response, highlighting the possibility that some differences may exist between the two loci.

Conclusions

Canonical and non-canonical JAK/STAT genes may regulate distinct and overlapping sets of genes and may perform specific overall functions in physiology. Further studies at different developmental stages, or using distinct tissues may identify additional targets and provide insight into which gene targets are unique to the canonical or non-canonical pathway.

The histone lysine demethylase 4 (Kdm4/Jmjd2/Jhdm3) family is highly conserved across species and reverses di- and tri-methylation of histone H3 lysine 9 (H3K9) and lysine 36 (H3K36) at the N-terminal tail of the core histone H3 in various metazoan species including Drosophila, C.elegans, zebrafish, mice and humans. Previous studies have shown that the Kdm4 family plays a wide variety of important biological roles in different species, including development, oncogenesis and longevity by regulating transcription, DNA damage response and apoptosis. Only two functional Kdm4 family members have been identified in Drosophila, compared to five in mammals, thus providing a simple model system. Drosophila Kdm4 loss-of-function mutants do not survive past the early 2nd instar larvae stage and display a molting defect phenotype associated with deregulated ecdysone hormone receptor signaling. To further characterize and identify additional targets of Kdm4, we employed a genome-wide approach to investigate transcriptome alterations in Kdm4 mutants versus wild-type during early development. We found evidence of increased deregulated transcripts, presumably associated with a progressive accumulation of H3K9 and H3K36 methylation through development. Gene ontology analyses found significant enrichment of terms related to the ecdysteroid hormone signaling pathway important in development, as expected, and additionally previously unidentified potential targets that warrant further investigation. Since Kdm4 is highly conserved across species, our results may be applicable more widely to other organisms and our genome-wide dataset may serve as a useful resource for further studies.

The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone demethylases has been implicated in development and tumorigenesis. Here we show that Drosophila Kdm4A and Kdm4B are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. We further show that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. Our studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters.

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.