Uncovering Tip60's chromodomain specific RNA-binding role in neuronal function

Christina M. Thomas

doi:10.17918/00011179

Alzheimer's disease (AD) is a prominent neurodegenerative disorder that arises from a complex interplay of genetics, aging, and environmental factors that influence epigenetic maintenance of neuronal gene regulation. Through many studies, AD has been shown to have reduce histone acetylation sites with an accompanying reduction in transcription and improper alternative splicing events of specific neuronal target genes. In healthy brains, histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain epigenetic homeostasis of acetylation of histone tails through the addition and removal of acetyl moieties, respectively. This leads to specific neuronal gene control through regulation of transcription and alternative splicing events. Yet, within AD pathology, this balance is disrupted by a reduction of prominent HATs such as Tip60 and an increase in HDACs like HDAC2 that cause chromatin packaging alterations that suppresses neuronal genes that are essential for cognitive function. Moreso, alternative RNA splicing has been highlighted as a widespread hallmark in Alzheimer's disease. Our lab has previously identified Tip60 to be neuroprotective within a Drosophila AD model. In these studies, reduction of Tip60 protein levels were seen with a significant reduction in acetylation of histone marks of specific neuroplasticity genes that were reversed through increasing Tip60 levels. Further analysis by our lab highlighted that Tip60 had the ability to also bind to RNA molecules and that this RNA-binding interactions was disrupted within an AD model with an accompanying alteration in alternative splicing outcomes. While this RNA binding function of Tip60 has been shown to occur, it has not yet been revealed which domain of Tip60's protein is orchestrating this function. Here, we report Tip60's chromodomain for the first time as being the main domain of Tip60 responsible for its RNA binding function. Using high resolution homology to model Tip60's chromodomain, we uncover four amino acids residues that are fully conserved within its ortholog ESA1 that has already been shown to interact with RNA. These four amino acids, Tyr57, Tyr60, Asn64, and Arg66, are all polar, suggesting that they interact with RNA via hydrogen bonding. Specifically, Arg66 has a positive charge and is positioned within Tip60's predicted RNA binding loop, indicating that it could interact with RNA through complementation of its negative charge and easy accessibility. Here, we utilize a point mutation method within Drosophila to change Tyr57 and Arg66 to alanine to interrupt their predicted RNA binding function both singularly and together to create transgenic Tip60RNAmut. Inducible expression of these Tip60RNAmut transgenes in the nervous system are predicated to cause a dominant negative phenotype as Tip60 should still be recruited to its chromatin gene targets and form multi-protein complexes but should be unable to interact with RNA. We find that these specific mutations severely affect published alternative splicing outcomes of known genes that are implicated with AD pathology. Further, we reveal that these lines have a reduction in pupation and eclosion with an accompanying decrease in longevity. Functionally, these flies have a significant decrease in their negative geotaxis ability. Overall, this highlights Tip60's chromodomain has being the main domain responsible for its RNA binding function and influence on alternative splicing. Our newly developed novel transgenic Tip60RNAmut fly models should serve as powerful tools to tease apart neural functions dependent upon Tip60 RNA binding.

Uncovering Tip60's chromodomain specific RNA-binding role in neuronal function

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Uncovering Tip60's chromodomain specific RNA-binding role in neuronal function

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