Rubio, M. | Search bioRxiv

bioRxiv2018

Comparative RNA-seq based transcriptomic analysis of Aspergillus nidulans recombinant strains overproducing heterologous glycoside hydrolases

Filamentous fungi are important cellular factories for the production and secretion of homologous and heterologous enzymes such as carbohydrate-active enzymes. However, the regulation of protein secretion in these microorganisms requires more profound studies since the enzyme levels produced are usually below the levels required by industry for profitable processes. Genomic and transcriptomic approaches have been used to understand the overexpression and production of heterologous enzymes and their capacity to induce different cellular biological processes. To investigate this regulation, Aspergillus nidulans recombinant strains were analyzed by transcriptomics. We designed three A. nidulans recombinant strains producing the following heterologous proteins: alpha-arabinofuranosidase (AbfA), beta-glucosidase (BglC) and thermophilic mannanase (Tp-Man5). The heterologous genes abfA and bglC were highly expressed, while tp-man5 mRNA levels were low and similar to those of a reference gene. There was an indirect relationship between mRNA and protein secretion levels, suggesting that transcription is not a bottleneck for target gene expression in this system. Despite the distinct features of the recombinant proteins, 30 differentially expressed genes were common to all the recombinant strains, suggesting that these genes represent a general response to the expression of heterologous genes. We also showed that the early activation of the canonical unfolded protein response (UPR) pathway by hacA alternative splicing was normalized after 8h, except in the strain expressing BglC, suggesting either no accumulation of the BglC misfolded form or the presence of an alternative endoplasmic reticulum (ER) stress and UPR pathway. Finally, to focus our analysis on the secretion pathway, a set of 374 genes was further evaluated. Seventeen genes were common to all the recombinant strains, suggesting again that these genes represent a general response of A. nidulans cells to the overexpression of recombinant genes, even thermophilic genes. Additionally, we reported the possible genetic interactions of these 17 genes based on coexpression network calculations. Interestingly, protein improvements are nongeneric, and improvements in the production of one target protein are not necessarily transferable to another one. Thus, this study may provide genetic and cellular background and targets for genetic manipulation to improve protein secretion by A. nidulans.

microbiology↗

bioRxiv2017

Comparative analysis of neutrophil and monocyte epigenomes

Neutrophils and monocytes provide a first line of defense against infections as part of the innate immune system. Here we report the integrated analysis of transcriptomic and epigenetic landscapes for circulating monocytes and neutrophils with the aim to enable downstream interpretation and functional validation of key regulatory elements in health and disease. We collected RNA-seq data, ChIP-seq of six histone modifications and of DNA methylation by bisulfite sequencing at base pair resolution from up to 6 individuals per cell type. Chromatin segmentation analyses suggested that monocytes have a higher number of cell-specific enhancer regions (4-fold) compared to neutrophils. This highly plastic epigenome is likely indicative of the greater differentiation potential of monocytes into macrophages, dendritic cells and osteoclasts. In contrast, most of the neutrophil-specific features tend to be characterized by repressed chromatin, reflective of their status as terminally differentiated cells. Enhancers were the regions where most of differences in DNA methylation between cells were observed, with monocyte-specific enhancers being generally hypomethylated. Monocytes show a substantially higher gene expression levels than neutrophils, in line with epigenomic analysis revealing that gene more active elements in monocytes. Our analyses suggest that the overexpression of c-Myc in monocytes and its binding to monocyte-specific enhancers could be an important contributor to these differences. Altogether, our study provides a comprehensive epigenetic chart of chromatin states in primary human neutrophils and monocytes, thus providing a valuable resource for studying the regulation of the human innate immune system.

genomics↗