pyMS-Vis, an Open-Source Python Application for Visualizing and Investigating Deconvoluted Top-Down Mass Spectrometric Experiments: A Histone Proteoform Case Study
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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Engineering topics
Umen, James G.
Publications and source records attributed to Umen, James G..
The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle
Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. Here, we interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G 1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G 0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.
59 BASIC BIOLOGICAL SCIENCES↗
An Improved Top-Down Mass Spectrometry Characterization of Chlamydomonas reinhardtii Histones and Their Post-translational Modifications
We present an updated analysis of the linker and core histone proteins and their proteoforms in the green microalga Chlamydomonas reinhardtii by top-down mass spectrometry (TDMS). The combination of high-resolution liquid chromatographic separation, robust fragmentation high, mass spectral resolution, the application of a custom search algorithm, and extensive manual analysis enabled the characterization of 86 proteoforms across all four core histones H2A, H2B, H3 and H4, and the linker histone H1. All canonical H2A paralogs, which vary in their C-termini, were identified, along with an unreported non-canonical variant that had both high levels of acetylation and C-terminal truncations. Similarly, a majority of the canonical H2B paralogs were identified, along with a smaller non-canonical variant, H2B.v, that was highly acetylated. Histone H4 exhibited a novel acetylation profile that differs significantly from that found in other organisms. A majority of H3 was monomethylated at K4, while a small fraction of H3 was trimethylated at K4 and also highly acetylated.
59 BASIC BIOLOGICAL SCIENCES↗