U of Pennsylvania Garcia Lab - dictyostelium_histone_ptms

Role of Epigenetics in Unicellular to Multicellular Transition in Dictyostelium

  • Organism: Dictyostelium discoideum AX2
  • Instrument: Q Exactive HF,Orbitrap Fusion
  • SpikeIn: No
  • Keywords: epigenetics, histone, multicellularity
  • Lab head: Lindsay Pino
Abstract
The evolution of multicellularity is a critical event that remains incompletely understood. We use the social amoeba, Dictyostelium discoideum, one of the rare organisms that exists in both unicellular and multicellular stages, to examine the role of epigenetics in regulating multicellularity. While transitioning to multicellular states, patterns of H3K4 methylation and H3K27 acetylation significantly change. By combining transcriptomics, epigenomics, chromatin accessibility, and syntenic analyses with other unicellular and multicellular organisms, we identify 52 conserved genes, which are specifically accessible and expressed during multicellular states. We validated that four of these genes, including the H3K27 deacetylase hdaD, are necessary and that an SMC-like gene, smcl1, is sufficient for multicellularity. These results highlight the importance of epigenetics in reorganizing chromatin architecture to facilitate the evolution of multicellularity.
Experiment Description
Orbitrap liquid chromatography-mass spectrometry. Peptides were analyzed with a Thermo Dionex UPLC coupled with a Thermo Q-Exactive HF tandem mass spectrometer. We used an in-house pulled column created from 75 μm inner diameter fused silica capillary packed with 3 μm ReproSil-Pur C18 beads (Dr. Maisch) to 30 cm. Solvent A was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 80% acetonitrile. For each injection, we loaded approximately 1 μg peptides and separated them using a 47-minute gradient from 2 to 35% B, followed by a 13 min washing gradient. For data dependent acquisition (DDA) analysis, a Top20 method was used (default charge state 2, minimum AGC target 5e4, charge exclusion 1 and >6, and dynamic exclusion 4s) with full MS (resolution 120,000; AGC 3e6, maximum IT 50 ms) and data dependent-MS2 (resolution 15,000; AGC 1e6, max IT 40 ms, isolation window 2.0 m/z, NCE 27)). For data independent acquisition (DIA) analysis, the Thermo Q-Exactive HF was configured to acquire 25 × 24 m/z (covering 300-900 m/z) precursor isolation window DIA spectra (30,000 resolution, AGC target 1e6, maximum inject time 60 ms, 27 NCE) using an optimized staggered window pattern. Precursor spectra (target range ± 15 m/z at 60,000 resolution, AGC target 1e6, maximum inject time 60 ms) were interspersed every 51 MS/MS spectra. The isolation window scheme is detailed in Supplemental Table 1. For parallel reaction monitoring (PRM), a Thermo Fusion tribrid mass spectrometer was configured to acquire target m/z using Orbitrap tMS2 (30,000 resolution, AGC target Standard, maximum inject time 60 ms, 30% CID CE) with precursor spectra (target range 290-910 m/z at 60,000 resolution, AGC target 1e6, maximum inject time 60 ms) interspersed every 50 MS/MS spectra. The isolation list is detailed in Supplemental Table 1. DDA data analysis. RAW files were converted to MZML using MSConvert (version 3.0.18). Byonic (version 3.10.2) was used to search a focused Dictyostelium FASTA with histone proteins and contaminants (Supplemental File 1) within Proteome Discoverer (version 2.4) using the following parameters: arg-c digestion, 2 max missed cleavages, minimum peptide length 7; 8 maximum modifications; fixed carbamidomethyl on C and U, variable oxidation on M, variable acetylation on protein N-terminus and K, methylation on R, dimethylation on K and R, trimethylation on protein N-terminal A and K, phosphorylation on S, T, and Y, propionylation on peptide N-terminus and K, propionyl-methylation on K, and GG-propionylation on K; precursor mass tolerance 10 ppm, product mass tolerance 20 ppm; HCD fragmentation. DIA data analysis. Mass spectrometry data files were demultiplexed and converted to MZML using MSConvert (version 3.0.18) (26). Database search results from DDA data analysis were built into a spectral library using Bibliospec (Frewen & MacCoss, 2007) in Skyline-daily (version 20.1) (MacLean et al., 2010). DIA files were imported into Skyline using MS/MS IDs in the spectral library (all settings default except Peptide Settings: digestion enzyme ArgC [R|P], background proteome from Uniprot Dictyostelium FASTA (accessed 21 Dec 2020, 12742 entries); Transition Settings: Filter for precursor charges 1-3, ion charges 1-2, ion types y, b, p; from ion 1 to last ion; Full-Scan MS1 filtering with isotope peaks included (Count) and MS/MS filtering for DIA acquisition, centroid product mass analyzer, and Results only isolation scheme; use only scans within 5 minutes of MS/MS IDs). PRM data analysis. A target list was built from the Cartesian product combination of known modifications and the Dictyostelium histone proteins (Uniprot, accessed 2020 Dec 31, 7 entries) using a custom Python script. Skyline-daily (version 20.1) was configured with these target peptides (all settings default except Peptide Settings: digestion enzyme ArgC [R|P], background proteome from Uniprot Dictyostelium histone FASTA (accessed 31 Dec 2020, 7 entries); Transition Settings: Filter for precursor charges 1-3, ion charges 1-2, ion types y, b, p; from ion 1 to last ion; Full-Scan MS1 filtering with isotope peaks included (Count) and MS/MS filtering for Targeted acquisition, centroid product mass analyzer; Include all matching scans). Data was imported and peaks were manually curated using isotope dot product, dot product when spectral library entries were available, and retention time information.
Sample Description
Strain. The Axenic strain (AX2) of Dictyostelium discoideum was generously given by Peter Devreotes at Johns Hopkins University. This stain was used as the main material for RNA-seq, ChIP-seq and ATAC-seq experiments. The other axenic strain (AX4) and the srfA knock out mutant was obtained from Dicty Stock Center. We cultured the strain at 22 °C shaken in the incubator at 180 rpm in HL5 medium with 300 μg/ml streptomycin. Histone chemical derivatization and peptide preparation for mass spectrometry. Histones were propionylated as previously described (Sidoli et al., 2016). Briefly, proteins were diluted with acetonitrile and neutralized with ammonium hydroxide. Chemical derivatization of lysines was performed with propionic anhydride twice, proteins were digested with trypsin, then two additional rounds of derivatization were performed. Propionylated peptides were desalted using an MCX protocol (Oasis MCX cartridge 1cc/30 mg LP, Waters Corporation).
Created on 2/17/21, 7:59 PM
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histone_garcia_v0-9-2_greer_onlyhistlibrary_2021-01-11_15-29-49.sky.zip2021-02-17 19:58:15101362386,13112