Mair, T. | Search bioRxiv

bioRxiv2021

Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

De novo mutations (DNMs) are an important player in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined [~]4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant frequencies of 10-4 to 10-5, with an overall mutation frequency of the region of [~]6x10-7. Some of the substitutions were re-current and were found at a higher variant frequency in older donors than in younger ones, or exclusively, in older donors. Also, older donors harbored more mutations associated with congenital disorders. Other mutations were present in both age groups suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independent of age, the frequency and deleteriousness of the mutational spectra was more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain-of-function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

genetics↗

bioRxiv2020

ATAC-seq identifies chromatin landscapes linked to the regulation of oxidative stress in the human fungal pathogen Candida albicans

Human fungal pathogens often encounter fungicidal stress conditions upon host invasion, but they can swiftly adapt by transcriptional reprogramming that enables pathogen survival. Fungal immune evasion is tightly connected to chromatin regulation. Hence, fungal chromatin modifiers pose alternative treatment options to combat fungal infections. Here, we present an ATAC-seq protocol adapted for the opportunistic pathogen Candida albicans to gain further insight into the interplay of chromatin accessibility and gene expression mounted during fungal adaptation to oxidative stress. The ATAC-seq workflow facilitates the robust detection of genomic regions with accessible chromatin, but also allows for the precise modeling of nucleosome positions in C. albcians. Importantly, the data reveal genes with altered chromatin accessibility in upstream regulatory regions, which correlate with transcriptional regulation during the oxidative stress response. Interestingly, many genes show increased chromatin accessibility yet no change in gene expression upon stress exposure. Such chromatin signatures could predict yet unknown regulatory factors under highly dynamic transcriptional control. In addition, de novo motif analysis in genomic regions with increased chromatin accessibility upon hydrogen peroxide treatment shows significant enrichment for Cap1 binding sites, a major factor of oxidative stress responses in C. albicans. Taken together, the ATAC-seq workflow enables the identification of chromatin signatures and uncovers the dynamics of regulatory mechanisms mediating environmental adaptation of C. albicans to host immune surveillance. ImportanceThe opportunistic fungal pathogen Candida albicans colonizes and infects various tissues and organs of the human host. This is due to its rapid environmental adaptation facilitated by changes in gene expression coupled to chromatin alterations. Recent advances in chromatin profiling approaches, such as the development of ATAC-seq, shed light on the dynamic interplay of chromatin accessibility and transcriptional control. The here presented expansion of the ATAC-seq method to C. albicans demonstrates the robustness of ATAC-seq to detect dynamic modulations of chromatin accessibility in response to oxidative stress. This work serves as a basis to further exploit this application to characterize regulatory mechanisms that drive fungal environmental adaptation, such as during host invasion, and thus, will open novel antifungal treatment strategies targeting fungal chromatin regulation.

microbiology↗