Changes in histone abundance and post-translational modifications in Rana [Lithobates] catesbeiana tissues during thyroid hormone-dependent metamorphosis.

Abstract

Amphibian metamorphosis is thyroid hormone (TH)-dependent and involves extensive gene expression changes in differentiated tissues that result in organismal remodeling. This is likely to involve changes in the epigenome that are poorly understood. We used tandem mass spectrometry (MS/MS) to investigate global variations in histone abundance and histone post-translational modifications (PTMs) during natural metamorphosis to identify putative epigenomic mediators of TH-mediated postembryonic development. Blood, liver, and tailfin from five Rana (Lithobates) catesbeiana (American bullfrog) individuals were collected from functionally athyroid premetamorphic tadpoles and from tadpoles at metamorphic climax (maximal TH levels). Only blood and liver were collected from five individual R. catesbeiana frogs (suprabasal TH levels), as the tailfin is resorbed by this stage. Nuclei were acid extracted to enrich for basic proteins, including histones and other chromatin-associated proteins. MS/MS data were queried against the R. catesbeiana proteome using open search parameters to capture modified histone peptides and stringent statistical evaluation was performed. The abundance of multiple histone variants and isoforms changed throughout metamorphosis. In particular, histone H1.0 increased over the metamorphic stages in all three tissue with a concurrent decrease in H1.3 in blood and liver. Further, H2A variants H2A.X and macroH2A.1 decreased over metamorphosis in blood while H2A type 2-C, H2A.Z-2 and macroH2A.2 increased in liver. We identified putative changes in histone PTMs by analyzing the abundance of peptides carrying mass shifts corresponding to methylation, acetylation and phosphorylation. Our findings include an increase in H1.3 mono- and dimethylation in blood and liver over metamorphosis and suggest a temporary increase of H3.2 phosphorylation at metamorphic climax in liver. To our knowledge, this is the first non-targeted analysis of histone isoform and variant abundance and PTMs during TH-mediated amphibian metamorphosis. Due to the conserved nature of TH signaling, our findings broaden our understanding of the coordinated variations in histone abundance and PTMs that occur across all histone families to direct the tissue-specific transitions that are critical for TH-driven development in amphibians and other vertebrate species.