Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain.

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species

Identyfikacja składników proteomu jądra komórkowego Arabidopsis thaliana i analiza modyfikacji potranslacyjnych histonu H1

Jądro jest jedną z najważniejszych, a zarazem, z powodu wyjątkowej złożoności, najsłabiej poznanych organelli komórki. Proteom jądra jest wysoce dynamiczny i, ze względu na liczne połączenia z cytoplazmą i stałą wymianę białek pomiędzy organellami, trudny do zdefiniowania. W literaturze brak kompleksowych badań proteomicznych jądra komórek roślinnych, w szczególności Arabidopsis, najważniejszego organizmu modelowego w biologii roślin. Przedstawione tu badania są pierwszą tego rodzaju analizą proteomu jądrowego Arabidopsis. Jądra użyte do analiz izolowano z zawiesinowej kultury komórek T-87 w sześciu powtórzeniach biologicznych za pomocą opracowanej w tym celu metody. Zastosowano podejście ‘shotgun proteomics’ polegające na trawieniu całego proteomu i późniejszej analizie mieszaniny peptydów za pomocą układu HPLC połączonego ze spektrometrem mas. Użycie spektrometru mas Orbitrap Velos pozwoliło na zastosowanie strategii ‘high‑high’, w której zarówno pomiary widm MS, jak i MS/MS przeprowadzano z wysoką rozdzielczością. Łącznie wykryto 6 753 białka jądrowe, będące produktami 4 862 genów. Dla prezentacji wyników doświadczeń utworzono stronę www działającą w oparciu o bazę danych pozwalającą na zmianę kryteriów identyfikacji białek. Aby stworzyć grupę odniesienia, przeanalizowano całościowy proteom komórek T‑87, co pozwoliło na identyfikację 6 342 białek, produktów 4 406 genów. Połowę z białek zidentyfikowanych w preparatach jądra komórkowego znaleziono w preparatach całych komórek. Wśród białek tych znajdowały się liczne białka typowe dla jądra, co sugeruje duży udział proteomu jądra w proteomie badanych komórek. Analizy „Gene Ontology” wykazały zasadnicze różnice klas białek wzbogaconych w obu badanych preparatach. Modyfikacje potranslacyjne histonów, jednego z głównych składników proteomu jądra komórkowego, są kluczowe dla mechanizmów regulacji ekspresji genów. Modyfikacje histonów rdzeniowych są intensywnie badane. Histon H1 jest białkiem znacznie słabiej poznanym, jego funkcja nie została do tej pory wyjaśniona. Wykonanie badań H1 Arabidopsis wymagało opracowania metody jego izolacji i rozdziału za pomocą HPLC, co doprowadziło do odkrycia, że stężenie nieallelicznego wariantu H1.2 jest 13‑to krotnie wyższe niż drugiego wariantu H1.1. Do tej pory przyjmowano, że stężenia H1.1 i H1.2 są zbliżone. Stwierdzono także występowanie mniejszego, nie wykrytego dotychczas produktu alternatywnego składania transkryptu H1.2. Modyfikacje potranslacyjne roślinnego histonu H1 nie były dotychczas badane. Przeprowadzone w niniejszej pracy analizy doprowadziły do wykrycia po raz pierwszy licznych modyfikacji potranslacyjnych takich, jak fosforylacja, acetylacja, krotonylacja, formylacja, mono- i di-metylacja oraz propionylacja. Dodatkowo, stwierdzono występowanie szeregu nieznanych modyfikacji oraz przyłączenia kationu żelaza II do N-końcowej domeny H1

Phylogeny-based systematization of arabidopsis proteins with histone H1 globular domain

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species

Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants

Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones

Chromatographic separation of total Arabidopsis H1.

<p>(A) Chromatogram. Masses and corresponding identification of H1 variants in different fractions are indicated on enlarged fragment of the chromatographic profile. (B) MALDI spectra of H1 variants.</p

Comparison of post-translational modifications in the GH1 domains of Arabidopsis H1.2, and human and mouse H1.3.

<p>(A) 3D models of the GH1 domains of <i>A</i>. <i>thaliana</i> H1.2, <i>H</i>. <i>sapiens</i> H1.3 and <i>M</i>. <i>musculus</i> H1.3. Residues subject to post-translational modification in Arabidopsis H1.2 identified in this study and those reported for human and mouse H1.3 by Wisniewski et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref016" target="_blank">16</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref036" target="_blank">36</a>] and Tan et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref044" target="_blank">44</a>] are shown in red. Colored dots denote the modifications according to the key. The models are shown in the same orientation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.g003" target="_blank">Fig 3E</a>. (B) Black cylinders and whiter arrows represent α-helices and β-turn, respectively. Multiple sequence alignment of the GH1 domains of Arabidopsis, human and mouse H1s. Residues modified post-translationally are highlighted using the same color scheme as in A.</p

Alignment of amino acid sequences of Arabidopsis H1.2 and H1.1 with identified post-translational modifications.

<p>The peptides with modifications identified by mass spectrometry are shown under the corresponding full sequence. Circles over the full sequence mark amino acids, with color filling the circle indicating the type of modification. For modifications marked “other” (white circle with dark green contour), the mass of modification in Da (eg. +99.000 Da) and the name of the possible chemical compound (eg. glycerophosphorylation) are indicated. The symbols: K, KV and KH correspond to lysine, lysine-valine and lysine-histidine, respectively. The color of letters corresponding to modified amino acids in the peptides corresponds to the type of modification, as shown for circles. Fragments of peptides identical in H1.2 and H1.1 are marked by italics. The sequences corresponding to globular domain (GH1) and S/TPxK motives are shaded in green and yellow, respectively. Sequence absent in a second splice variant of H1.2 is shaded in pink. Digestion sites by trypsin and trypsin and Arg-C proteases are marked by green and blue triangles, respectively. Green “n-” denotes acetylated protein N-terminus. Note that H1.2 and H1.1 lack initial methionine (amino acid 1) marked by grey “M” in the full sequence.</p

3D models of the GH1 domain of Arabidopsis H1.2 in complex with a nucleosome.

<p>(A) symmetric model of GH1-nucleosome complex from Syed et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref001" target="_blank">1</a>], (B) asymmetric model from Zhou et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref002" target="_blank">2</a>], (C) asymmetric model from Song et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147908#pone.0147908.ref003" target="_blank">3</a>]. The presented structures correspond to the respective H1-nucleosome models obtained from the authors, with the original GH1 replaced by the 3D model of the Arabidopsis H1.2 GH1 (blue). Schematic representations of GH1-mononucleosome and GH1-dinucleosome complexes are shown in the upper left corners. (D-F) Enlargement of GH1 binding with residues targeted by post-translational modifications shown in red. The identified modifications are denoted by colored dots: methylation—magenta; formylation—olive; acetylation—green; crotonylation—blue. The models in D, E and F are shown in the same orientation as those in A, B and C, respectively.</p

Comparison of post-translational modifications present in different regions of Arabidopsis and mammalian H1s.

<p>Comparison of post-translational modifications present in different regions of Arabidopsis and mammalian H1s.</p