Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins

Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9

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