Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development

Abstract

Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as gamma-irradiation altered histone acetylation patterns. Strikingly, gamma-irradiation dramatically increased acetylation at H3K18, a site linked to DNA repair via non-homologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system, and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.

Proteomic-based approaches to cardiac development and disease

“…It is therefore evident that research into the molecular roots of CHD should involve direct assessments of protein expression levels and interaction networks. Tandem mass spectrometry (MS/MS)-based analysis has proven invaluable in studying the temporal and spatial distribution of proteins during development in a range of animal model systems [19,[24][25][26][27][28][29]. This type of proteomic-based approach allows not only for the generation of a compendium of proteins of a given cell type at a given stage of development, but also provides information for the characterization of post-translational modifications (PTMs).…”

Quantification of the effect of site-specific histone acetylation on chromatin transcription rate

“…These lysines can be acetylated by histone acetyltransferases, and the number of acetylated residues correlates with RNA transcription activity (15,16,21). Among several H4 acetylation states (22)(23)(24)(25), tetra-acetylation at K5/K8/K12/K16 is particularly important, because this hyperacetylated state is found in euchromatin regions where the nearby genes are transcriptionally most active (26). H4 acetylation at K5/K8/K12/K16 correlates with the expression of both the RNAP II-and III-transcribed genes (15,16).…”

Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors

“…Given the limitations of antibody-based approaches (e.g., cross-reactivity and limited epitope accessibility), mass spectrometry (MS) – assisted approach providing highly standardized procedures for sample preparation, high resolution instrumentation and data analysis algorithms, is currently an important analytical tool to study post-translational modifications (PTMs; Aebersold and Mann, 2016 ). It has been extensively used for both qualitative and quantitative analyses of histone modifications, including phosphorylation, methylation and acetylation in many model species ( Drury et al, 2012 ; Tweedie-Cullen et al, 2012 ; Krejčí et al, 2015 ; Moraes et al, 2015 ; de Jesus et al, 2016 ; Henry et al, 2016 ), as reviewed by Zheng et al (2016) . MS also offers the ability to examine novel and combinatorial PTMs in a high-throughput fashion.…”