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Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

ABSTRACT: Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We sequenced Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non repressive euchromatic states. The T. brucei H2A C terminal tail is hyperacetylated, containing up to 5 acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in higher eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: JP Maree

LAB HEAD: Hugh George Patterton

PROVIDER: PXD024574 | Pride | 2022-08-21

REPOSITORIES: Pride

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			Action	DRS
	FUS04872_PF_1.dat	Other
	FUS04872_PF_1.raw	Raw
	FUS04873_PF_2.dat	Other
	FUS04873_PF_2.raw	Raw
	FUS04880_PF_3.dat	Other

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Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

Project description:Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We sequenced Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis‑histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non‑repressive euchromatic states. The T. brucei H2A C‑terminal tail is hyperacetylated, containing up to 5 acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in higher eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.

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Project description:The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nanoLC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

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Project description:During DNA replication parental, nucleosomic histones are segregated almost symmetrically to the two daughter DNA strands enabling mitotic inheritance of histones and their PTMs. In MCM2 histone-binding mutant ESCs (MCM2-2A), histones are recycled asymmetrically to the leading strand. To evaluate if loss of parental histones contributes to the asymmetry, we tracked new and old histones quantitatively by pulse-Triple-SILAC-MS. The quantification showed no change in dilution and incorporation dynamics in MCM2-2A cells, demonstrating that parental histones are not lost at replication forks but re-routed from the lagging to the leading strand.

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Systematic genetic and proteomic screens identify yeast H2BK37 and mammalian H2BK34 methylation as an evolutionary conserved meiotic mark

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Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

Project description:Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

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