Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.

Co-Expression Networks in Chlamydomonas Reveal Significant Rhythmicity in Batch Cultures and Empower Gene Function Discovery

The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.

The Expression Potential of New Yeast Strains of the Komagataella Genus

The expression potential of various strains from the Collection of the National Bio-Resource Center (BRC VKPM) collection belonging to the species Komagataella kurtzmanii, K. phaffii, K. mondaviorum has been assessed by the level of production of the heterologous enzyme Citrobacter freundii phytase. Heterologous expression in the K. mondaviorum strains was observed for the first time. The strains of K. phaffii Y-4288, K. mondaviorum Y-4331 and K. phaffii Y-4287 were identified with a high level production of the heterologous enzyme, a high growth rate, the ability to accumulate a large amount of biomass and moderate thermotolerance. It was shown that the average productivity of the transformants based on K. phaffii Y-4288, K. mondaviorum Y-4331, and K. phaffii Y-4287 strains exceeds that of the commercial industrial recipient strain K. phaffii GS115 Y-2837 by more than 3, 5 and 6 times, respectively. The K. phaffii Y-4287 and K. mondaviorum Y-4331 strains exhibited moderate thermotolerance and the ability to accumulate a heterologous product at 37° C. The high expression potential of the identified strains opens up the possibility of creating recipient strains on their basis for high-level production of heterologous proteins. Key words: Komagataella, Pichia pastoris, thermotolerance, expression of heterologous proteins Funding - This work was supported by the Ministry of Science and Higher Education of the Russian Federation (grant no. 075-15-2019-1658 dated October 31, 2019) and was carried out using the resources of the Unique Scientific Facility of the "All-Russian Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»---GOSNIIGENETIKA.

Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.

Co-Expression Networks in the Green Alga Chlamydomonas reinhardtii Empower Gene Discovery and Functional Exploration

ABSTRACTThe unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis, cilium assembly and function, lipid and starch metabolism and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes from manually-curated and community-generated gene lists. We extracted 400 high-confidence cilia-related genes at the intersection of multiple co-expressed lists, illustrating the power of our simple method. Surprisingly, Chlamydomonas experiments did not cluster according to an obvious pattern, suggesting an underappreciated variable during sample collection. One possible source of variation may stem from the strong clustering of nuclear genes as a function of their diurnal phase, even in samples collected in constant conditions, indicating substantial residual synchronization in batch cultures. We provide a step-by-step guide into the analysis of co-expression across Chlamydomonas transcriptome datasets to help foster gene function discovery.One-sentence summarywe reveal co-expression potential between Chlamydomonas genes and describe strong synchronization of cells grown in batch cultures as a possible source of underappreciated variation.

Genome-wide chromatin accessibility is restricted by ANP32E

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.

Genome-wide Chromatin Accessibility is Restricted by ANP32E

ABSTRACTGenome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how control of genomic H2A.Z localization by ANP32E contributes to chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that through antagonism of H2A.Z, ANP32E restricts genome-wide DNA access. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.