Biology subjects

Kobayashi, W.

Publications and source records attributed to Kobayashi, W..

4 recordsLinked to original sources

bioRxiv2023

Nucleosome-bound NR5A2 structure reveals pioneer factor mechanism by minor groove anchor competition

Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors, which bind nucleosomal DNA and initiate gene expression from closed chromatin. How pioneer factors perform this function is poorly understood. Nr5a2 is a pioneer factor that is critical for zygotic genome activation (ZGA) in totipotent embryos, pluripotency in embryonic stem cells and metabolic regulation in adult tissues. To study how Nr5a2 functions as a pioneer factor, we used cryo-electron microscopy to determine a structure of human NR5A2 bound to a nucleosome. The structure shows that the conserved C-terminal extension (CTE) loop of the NR5A2 DNA-binding domain competes with a DNA minor groove anchor of the nucleosome and releases entry-exit site DNA at a [~]50{degrees} angle. Residue aspartic acid 159 of the CTE loop induces the rearrangement by shifting a histone H2A 2 helix towards the nucleosome center. Mutational analysis showed that NR5A2 D159 is largely dispensable for DNA binding but required for stable nucleosome association and persistent DNA unwrapping. These findings suggest that NR5A2 belongs to a previously unknown class of pioneer factors that can use minor groove anchor competition to destabilize nucleosomes and facilitate gene expression changes during reprogramming.

biochemistry↗

bioRxiv2023

A chromosome-level genome assembly of amodel conifer plant, the Japanese cedar,Cryptomeria japonica D. Don

Japanese cedar (Cryptomeria japonica D. Don) is the most important Japanese forest tree, occupying about 44% of artificial forests in Japan, and planted in East Asia, Azores Archipelago, and some islands in the Indian Ocean. Although the huge genome of the species (ca. 11 Gb) with abundant repeat elements might have been an obstacle for genetic analysis, the species is easily propagated by cutting, flowered by plant hormones like gibberellic acid, transformed by agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica are preferable to make the species a model conifer for which reference genome sequences are necessary. In this study, we report the first chromosome-level assembly for C. japonica (2n = 22) using a third generation selfed progeny with an estimated homozygosity of 0.96. Young leaf tissue was used to extract high-molecular-weight DNA (>50 kb) for HiFi PacBio long read sequencing and to construct Hi-C/Omni-C library for Illumina short read sequencing. Using the 29x and 26x genome coverage of HiFi and Illumina reads, respectively, de novo assembly resulted in 2,650 contigs (9.1 Gb in total) with N50 contig size of 12.0 Mb. The Hi-C analysis mapped 97% of the nucleotides on the 11 chromosomes. The assembly was verified by comparing with a consensus linkage map of 7,785 markers. The BUSCO analysis confirmed ~91% of conserved genes. Annotations of genes, repeat elements and synteny with other Cupressaceae and Pinaceae species were performed, providing fundamental resources for genomic research of conifers.

genomics↗

bioRxiv2022

Zygotic genome activation by the totipotency pioneer factor Nr5a2

Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA) in totipotent embryos. Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. Based on de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse embryos. Nr5a2 binds to its motif within a subtype of SINE B1/Alu transposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear family receptors. Consistent with a role in ZGA, Nr5a2 is required for progression beyond the 2-cell stage. Nr5a2 promotes chromatin accessibility during ZGA and binds to entry/exit sites of nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that distinctly regulates totipotency and pluripotency during mammalian development. One-Sentence SummaryNr5a2 is an essential pioneer transcription factor that activates expression of zygotic genes in mouse embryos.

developmental biology↗

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗