NAxtra magnetic nanoparticles for low-cost, efficient isolation of mammalian DNA and RNA.

A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low- and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.

Optimized synthesis of polyacrylic acid-coated magnetic nanoparticles for high-efficiency DNA isolation and size selection.

Solid-phase reversible immobilization (SPRI) bead technology is widely used in molecular biology for convenient DNA manipulation. However, commercial SPRI bead kits lack cost advantages and flexibility. It is, therefore, necessary to develop new and alternative cost-effective methods of on-par or better quality. Herein, an easy and cost-effective method is proposed for synthesizing polyacrylic acid-coated iron oxide nanoparticles (PAA-IONPs) through in situ polymerization at lab scale for high-efficiency nucleic acid extraction and size selection. A design of experiment (DoE) approach was used to investigate the influence of iron oxide nanoparticles (IONPs), acrylic acid (AA) monomer, and sodium dodecyl sulfate (SDS) surfactant amounts on the sizes and carboxyl group densities of PAA-IONPs. Thorough characterization by thermogravimetric analysis (TGA), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and vibrating sample magnetometry (VSM) highlights the importance of a low starting pH achieved by a high ratio of AA/IONPs, to yield the largest sizes (554 nm) and highest carboxyl group densities (2.13 mmol g-1) obtained in this study. An efficient DNA purification strategy is then presented using homemade beads-suspension buffer and optimized bead concentrations (17% PEG 8000, 2.5 M NaCl, and 3 mg mL-1 PAA-IONPs). This method shows comparable performance to the control (AMPure XP beads) for DNA recovery. An adjustable PAA-IONPs DNA purification system was also developed to be used for DNA-size selection at low DNA amounts (50-100 ng) with a high degree of resolution and recovery. In conclusion, this work offers an optimized PAA-IONPs synthesis protocol and a flexible DNA purification approach that will enable researchers to manipulate DNA under various conditions, holding the significant potential to benefit future molecular biology research and diagnostics.

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.

Going low to reach high: Small-scale ChIP-seq maps new terrain.

Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin-associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole-genome high-throughput sequencing (ChIP-seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP-seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small-scale ChIP-seq and analyzing data; and (c) the potential of small-scale ChIP-seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.

Histone Methylations Define Neural Stem/Progenitor Cell Subtypes in the Mouse Subventricular Zone.

Neural stem/progenitor cells (NSPCs) persist in the mammalian brain throughout life and can be activated in response to the physiological and pathophysiological stimuli. Epigenetic reprogramming of NPSC represents a novel strategy for enhancing the intrinsic potential of the brain to regenerate after brain injury. Therefore, defining the epigenetic features of NSPCs is important for developing epigenetic therapies for targeted reprogramming of NSPCs to rescue neurologic function after injury. In this study, we aimed at defining different subtypes of NSPCs by individual histone methylations. We found the three histone marks, histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 trimethylation (H3K27me3), and histone H3 lysine 36 trimethylation (H3K36me3), to nicely and dynamically portray individual cell types during neurodevelopment. First, we found all three marks co-stained with NSPC marker SOX2 in mouse subventricular zone. Then, CD133, Id1, Mash1, and DCX immunostaining were used to define NSPC subtypes. Type E/B, B/C, and C/A cells showed high levels of H3K27me3, H3K36me3, and H3K4me3, respectively. Our results reveal defined histone methylations of NSPC subtypes supporting that epigenetic regulation is critical for neurogenesis and for maintaining NSPCs.

5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication.

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.