Histone H3 Phosphorylation: Universal Code or Lineage Specific Dialects?

Post-translational modifications of histones modulate the functional landscape of chromatin and impinge on many DNA-mediated processes. Phosphorylation of histone H3 plays a role in the regulation of gene expression and in chromosome condensation/segregation. Certain evolutionarily conserved residues on histone H3—namely Thr3, Ser10, Thr11, and Ser28—are phosphorylated during interphase or mitosis in both metazoa and plants. However, many of the kinases involved in these events appear to have evolved independently in different lineages. Likewise, the mechanistic function of specific phosphorylated amino acids, although poorly understood, also seems to differ among eukaryotes. Moreover, some modifications, such as phosphorylation of histone H3 Ser10, appear to have both a positive and a negative connotation and only become meaningful in combination with other histone marks within a particular chromatin context. Thus, a detailed understanding of the influence of histone H3 phosphorylation on biological processes may require learning organismal dialects of the histone code

Origin of the polycomb repressive complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas

Polycomb group proteins play an essential role in the maintenance of cell identity and the regulation of development in both animals and plants. The Polycomb Repressive Complex 2 (PRC2) is involved in the establishment of transcriptionally silent chromatin states, in part through its ability to methylate lysine 27 of histone H3 by the Enhancer of zeste [E(z)] subunit. The absence of PRC2 in unicellular model fungi and its function in the repression of genes vital for the development of higher eukaryotes led to the proposal that this complex may have evolved together with the emergence of multicellularity. However, we report here on the widespread presence of PRC2 core subunits in unicellular eukaryotes from the Opisthokonta, Chromalveolata and Archaeplastida supergroups. To gain insight on the role of PRC2 in single celled organisms, we characterized an E(z) homolog, EZH, in the green alga Chlamydomonas reinhardtii. RNAi-mediated suppression of EZH led to defects in the silencing of transgenes and retrotransposons as well as to a global increase in histone post-translational modifications associated with transcriptional activity, such as trimethylation of histone H3 lysine 4 and acetylation of histone H4. On the basis of the parsimony principle, our findings suggest that PRC2 appeared early in eukaryotic evolution, even perhaps in the last unicellular common ancestor of eukaryotes. One of the ancestral roles of PCR2 may have been in defense responses against intragenomic parasites such as transposable elements, prior to being co-opted for lineage specific functions like developmental regulation in multicellular eukaryotes

Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e and \u3ci\u3eCoccomyxa\u3c/i\u3e sp. C-169

Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and carbohydrates, and the environmental factors influencing their accumulation is necessary to realize the full potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accumulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress in medium containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under strictly photoautotrophic conditions. Interestingly, nutrient depletion triggered a similar pattern of early synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green microalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in ribosomal polypeptides and some key enzymes for CO2 assimilation like ribulose-1,5-bisphosphate carboxylase/oxygenase. These results suggest that turnover of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, several genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engineering of algal strains with improved capacity to convert their biomass into useful biofuel precursors

Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing

Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER-LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga

Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane

<div><p>Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is “written” in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.</p></div

Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.

<p>(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.</p

Serine/threonine O-acetylation in sugarcane histone H3.

<p>(A) MS/MS spectrum of the [M+2H]²⁺ ion (<i>m</i>/<i>z</i> 556.3089) that matched the histone H3 peptide prKSacTGGKprAPR (residues 9–17) where S10 is acetylated. (B) MS/MS spectra of the doubly-charged precursor ion at <i>m</i>/<i>z</i> 598.8534 corresponding to H3T22 acetylation in the H3 peptide prKQLATacKprAAR (residues 18–26). Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.</p

Distribution patterns of histone post-translational modifications in sugarcane.

<p>(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.</p

Post-translational modifications identified in sugarcane histone Ss-H3.1 and Ss_H3.3 variant.

<p>Amino acid residues covered by the peptides identified by the MS/MS analysis are indicated in red. The modification sites identified are shown on top of the sequence and the amino acid residues highlighted in blue (lysine), green (arginine), brown (serine), purple (threonine) and light blue (tyrosine). The first amino acid methionine was omitted from the sequence.</p

Lysine acetylation in sugarcane histone H3.

<p>(A) MS/MS spectrum of the doubly-charged ion at <i>m</i>/<i>z</i> 570.8407 corresponding to the H3 peptide prKacQLATKprAAR (residues 18–26) where K18 is acetylated. (B) Fragment ions of the recorded in MS/MS spectrum for the [M+2H]²⁺ ion (<i>m</i>/<i>z</i> 563.8325) matches to the peptide prKacQLATKacAAR acetylated at positions K18 and K23. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.</p