Jurkowska, R. Z. | Search bioRxiv

bioRxiv2020

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

genetics↗

bioRxiv2020

Versatile workflow for cell type resolved transcriptional and epigenetic profiles from cryopreserved human lung.

The complexity of the lung microenvironment together with changes in cellular composition during disease progression make it exceptionally hard to understand the molecular mechanisms leading to the development of chronic lung diseases. Although recent advances in cell type resolved and single-cell sequencing approaches hold great promise for studying complex diseases, their implementation greatly relies on local access to fresh tissue, as traditional methods to process and store tissue do not allow viable cell isolation. To overcome these hurdles, we developed a novel, versatile workflow that allows long-term storage of human lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with next generation sequencing-based profiling, including single-cell approaches. We demonstrate that cryopreservation is suitable for isolation of multiple cell types from different lung locations and is applicable to both healthy and diseased tissue, including COPD and tumor samples. Basal cells isolated from cryopreserved airways retain the ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing (RNA-seq) and Illumina EPIC Array, we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of human lung cells. In addition, we obtained high-quality single-cell RNA sequencing data of cells isolated from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to human tissue.

cell biology↗