Biology subjects

Strenkert, D.

Publications and source records attributed to Strenkert, D..

5 recordsLinked to original sources

bioRxiv2021

An optimized ChIP-Seq framework for profiling of histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin Immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein crosslinking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

genomics↗

bioRxiv2021

Genome wide profiling of histone H3 lysine 4 methylation during the Chlamydomonas cell cycle reveals stable and dynamic properties of lysine 4 trimethylation at gene promoters and near ubiquitous lysine 4 monomethylation

Chromatin modifications are key epigenetic regulatory features with roles in various cellular events, yet histone mark identification, gene wide distribution and relationship to gene expression remains understudied in green algae. Histone lysine methylation is regarded as an active chromatin mark in many organisms, and is implicated in mediating active euchromatin. We interrogated the genome wide distribution pattern of mono- and trimethylated H3K4 using Chromatin-Immunoprecipitation followed by deep-sequencing (ChIP-Seq) during key phases of the Chlamydomonas cell cycle: early G1 phase (ZT1) when cells initiate biomass accumulation, S/M phase (ZT13) when cells are undergoing DNA replication and mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at TSSs of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at one or more time points during the cell cycle but not necessarily by continuous active transcription. This finding even applied to early zygotic genes whose expression may be dormant for hundreds or thousands of generations between sexual cycles; but core meiotic genes were completely missing H3K4me3 peaks at their TSS. In addition, bi-directional promoters regulating expression of replication dependent histone genes, had transient H3K4me3 peaks that were present only during S/M phase when their expression peaked. In agreement with biochemical studies, mono-methylated H3K4 was the default state for the vast majority of histones that were outside of TSS and terminator regions of genes. A small fraction of the genome which was depleted of any H3 lysine methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Genome wide H3K4me3 ChIP-Seq data will be a valuable resource, facilitating gene structural annotation, as exemplified by our validation of hundreds of long non-coding RNA genes.

genomics↗

bioRxiv2021

Chlamydomonas ATX1 is essential for Cu distribution towards the secretory pathway and maintenance of biomass in conditions demanding cupro-enzyme dependent metabolic pathways

Copper (Cu) chaperones, of which yeast ATX1 is a prototype, are small proteins with a Cu(I) binding Mx-CxxC motif, and are responsible for directing intracellular Cu towards specific client protein targets that use Cu as a cofactor. The Chlamydomonas reinhardtii ATX1 (CrATX1) was identified because of its high sequence similarity with yeast ATX1. Like the yeast homologue, CrATX1 accumulates in iron-deficient cells (but is not impacted by other metal-deficiencies), and YFP-ATX1 is distributed in the cytoplasm. Reverse genetic analysis using artificial microRNA (amiRNA) to generate lines with reduced CrATX1 abundance and CRISPR/CPF1 to generate ATX1 knock out lines validated a function for ATX1 in iron-poor cells, most likely because of an impact on metalation of the multicopper oxidase FOX1, which is an important component in high-affinity iron uptake. A more general impact on the secretory pathway is indicated by reduced growth of ATX1 mutant lines on guanine as a sole nitrogen source, which we attribute to loss of function of UOX1, a urate oxidase involved in guanine assimilation. The block of Cu trafficking towards the secretory pathway in ATX1 mutants is strikingly evident by a reduced amount of intracellular Cu in all conditions probed in this work.

biochemistry↗

bioRxiv2021

Single-cell Visualization and Quantification of Trace Metals in Chlamydomonas Lysosome-Related Organelles

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass-spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared to MS-based imaging techniques, XFM offers the advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii, to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal over-accumulation (Fe, Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions, and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells. Significance statementTransition metals are of crucial importance for primary productivity; their scarcity limits crop yield in agriculture and carbon sequestration at global scale. Copper (Cu), iron (Fe) and manganese (Mn) are among the most important trace elements that enable the redox chemistry in oxygenic photosynthesis. The single-celled, eukaryotic green alga Chlamydomonas reinhardtii is a choice experimental system for studying trace metal homeostasis in the context of phototrophy, offering all the advantages of a classical microbial system with a well-characterized photosystem and trace metal metabolism machinery of relevance to plants. This project identifies and differentiates different trace metal storage sites in Chlamydomonas and uncovers the dynamics of trace metal storage and mobilization in situations of fluctuating resources.

plant biology↗

bioRxiv2020

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons — termed TOC and TIC — located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56 and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TicToc), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and the here newly identified TicToc components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TicToc complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TicToc supercomplex in maintaining chloroplast proteostasis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗