DNA recombination techniques in mammalian cells has been applied to the production of therapeutic proteins for several decades. To be used for commercial production, established cell lines should stably express target proteins with high productivity and acceptable quality for human use. In the conventional transfection method, the screening process is laborious and time-consuming since superior cell lines had to be selected from an enormous number of transfected cell pools and clonal cell lines with a wide variety of transgene insertion locations. In this study, we demonstrated that the combination of a Tol2 transposon system and cell selection by cycloheximide resistance is an efficient method to express therapeutic proteins, such as human antibody in suspension culture of Chinese hamster ovary cells. The resulting stable cell lines showed constant productivity and cell growth over long enough cultivation periods for recombinant protein production. We anticipate that this approach will prove widely applicable to protein production in research and development of pharmaceutical products.

Figure: Antibody productivity and cell growth of clonal cell lines 30-2-2, 30-2-10, 30-2-11 in the presence or absence of CHX selection. Antibody productivity (A, B and C), cell growth (D, E and F) and specific antibody productivity (pg/cell/day) (G, H and I) in 30-2-2, 30-2-10, 30-2-11 were measured in the presence (open circles) or absence (black circles) of CHX selection pressure.

The human genome chromatin can be classified into euchromatin and heterochromatin, which have high and low transcription activities, respectively. In the classical view, it was believed that euchromatin has an open and decondensed structure, while heterochromatin is highly condensed.

A research team led by Professor Kazuhiro Maeshima of Genome Dynamics Laboratory (NIG), including a graduate student (JSPS Research Fellow DC2) Shiori Iida, SOKENDAI graduate student Masa A. Shimazoe, Technical Stuff Sachiko Tamura, and Assistant Professor Satoru Ide have put forward the model that euchromatin essentially forms condensed domains with sizes ranging from 100-300 nm in diameter based on new evidence from genomics and advanced imaging studies. They discuss this novel view of euchromatin in the cell and how the revealed organization is relevant to genome functions.

Physically condensed domains provide a higher-order regulation of transcription, while the extended fiber loops do not. Condensed domains likely hinder the accessibility of large transcription complexes to their target sequence located in the inner core of chromatin domains.

This exclusion effect can repress unintended gene expression. Although such a core of condensed domains seems to be inaccessible, condensed domains have the liquid-like property, allowing small transcription factors to penetrate the core of domains. Such small transcription factors may help a target sequence to float up to the domain surface to be transcribed. The authors suggest that condensed chromatin domains can work as Lego blocks of mitotic chromosomes during cell division, which create a smoother process for chromosome assembly and disassembly.

This work was supported by JSPS Fellowship, JSPD grants (JP21H02453, JP22H05606, JP21H02535, JP20H05936, JP16H06279(PAGS)), JSPS Research Fellow (JP23KJ0996(DC2)), JST SPRING (JPMJSP2104).

Figure: Euchromatic condensed domains provide higher-order regulation of transcription by physically excluding large transcription complexes (green spheres) from the inner core of the domains. Liquid-like property inside a condensed domain gives a certain degree of accessibility for gene expression. Small transcription factors interact with the target gene (open green circle nucleosomes) inside the inner core and cause it to float up to the domain surface to be transcribed. The condensed chromatin domains can work as Lego blocks of mitotic chromosomes during cell division.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of Acrofacial Dysostosis, Cincinnati-type. The predominant phenotypes observed in the initial cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique (11 novel) heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology which supports the possibility of variant-specific phenotypic effects in patients. To further explore variant-specific effects in vivo, we used CRISPR/Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in patients were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice (Figure A). Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes death of the affected cells and resulting embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects and differential tissue-specific requirements that provide novel insights into the underlying pathogenesis of ribosomopathies.

Assistant Professor Satoru Ide and Professor Kazuhiro Maeshima of Genome Dynamics Laboratory performed in vitro assessment of POLR1A variants, which impact rRNA transcription and the nucleolar morphology in human culture cells expressing wild-type or mutant POLR1A. Using a machine learning algorithm called “wndchrm”, Ide and Maeshima found that nucleolar morphologies of the mutants (based on POLR1A foci) are somehow corelated with their rRNA transcription capacities or status (Figure B).

The part to which NIG contributed was supported by the Japan Society for the Promotion of Science (JSPS) grants (22H05606, 21H02535 to SI; 19H05273 ,20H05936 to KM) etc. The image analysis by the machine learning algorithm “wndchrm” was conducted in collaboration with Dr. Noriko Saito in The Cancer Institute of JFCR.

Figure: （A）Polr1a variant murine embryos demonstrate more hypoplastic craniofacial primordia at E12 (upper panel, right), a reduced cerebral cortex area at E12 (middle panel, right) and gross enlargement of heart at P0 (lower panel, right), compared to wild type (all the panels, left), respectively. (B) (upper panel) Localization of fluorescently labeled POLR1A (cellular nucleus, cyan and human POLR1A variants, magenta). (lower panel) Phylogeny shows the similarity of the nucleolar morphologies of wild type and nine POLR1A variants. POLR1A variants showing transcription upregulation, transcription downregulation and no transcriptional change are indicated in red, blue, and black, respectively.

Press release (In Japanese only)

To quantify the biases introduced during human gut microbiome studies, analyzing an artificial mock community as the reference microbiome is indispensable. However, there are still limited resources for a mock community which well represents the human gut microbiome. Here, we constructed a novel mock community comprising the type strains of 18 major bacterial species in the human gut and assessed the influence of experimental and bioinformatics procedures on the 16S rRNA gene and shotgun metagenomic sequencing. We found that DNA extraction methods greatly affected the DNA yields and taxonomic composition of sequenced reads, and that some of the commonly used primers for 16S rRNA genes were prone to underestimate the abundance of some gut commensal taxa such as Erysipelotrichia, Verrucomicrobiota and Methanobacteriota. Binning of the assembled contigs of shotgun metagenomic sequences by MetaBAT2 produced phylogenetically consistent, less-contaminated bins with varied completeness. The ensemble approach of multiple binning tools by MetaWRAP can improve completeness but sometimes increases the contamination rate. Our benchmark study provides an important foundation for the interpretation of human gut microbiome data by providing means for standardization among gut microbiome data obtained with different methodologies and will facilitate further development of analytical methods.

Source: Hiroshi Mori et al., DNA Research doi: 10.1093/dnares/dsad010

Shiori Iida with her course certificate.
She is wearing a T-shirt of Analytical and
Quantitative Light Microscopy 2023 with
an illustration of a lobster and a microscope.

Analytical and Quantitative Light Microscopy

Marine Biological Laboratory

Genome Dynamics Laboratory

Meiosis is a special cell division that halves the chromosome number in preparation for fertilization. In this process, the genomic information of the parents is shuffled to generate a new gene combination (meiotic recombination), which is inherited to the next generation. The meiotic recombination machinery is widely conserved in eukaryotes and has been extensively studied. On the other hand, the mechanisms by which germ cells transition to meiosis are diverse among species, and little is known particularly in plants.

In this review, we mainly focused on meiosis of flowering plants, and explained how the archesporial cells, which produce meiocytes in the future, develop and eventually achieve meiosis with the latest findings. We also highlight the findings of our research group that the callose polysaccharide, a special plant cell wall components, plays an essential role in regulating the proper initiation timing and progression of male meiosis.

The image using the figures in our review paper was selected for the cover story of Plants Vol. 12, No. 10, including 143 articles.

Figure: A model of callose synthesis and transport in anthers
(A) Cross sections of the anther just before meiosis. (Upper) Staining of cellulosic cell walls. (Bottom) Double immuno-staining of callose (red) and GSL5 (yellow). Bar=20µm. (B) The plasma membrane-anchored callose synthase (GSL) of male meiotic cells (PMCs) releases synthesized callose to apoplasts between PMCs (APOPLAST) just prior to meiosis transition. Callose migrates to the apoplast between PMCs and tapetum cells (TAPETUM) where GSL is absent, and eventually envelop all PMCs within the anther locule.

To proliferate, cells must replicate chromosomal DNA, which encodes the genetic information. MCM2–7, the replication helicase that unwinds a double-strand DNA to generate a single-strand DNA, is essential for chromosomal DNA replication. The replicative MCM2–7 helicase forms a ring-shaped hexamer.

This review paper was published in Biochemical Society Transactions, a review journal published by the Biochemical Society. The review discusses how the MCM2–7 hexamer is efficiently assembled in cells and its physiological significance based on our recent report. In this review, we proposed a model in which MCMBP functions as a chaperone to promote the assembly of the MCM2–7 hexamer, resulting in the supply of sufficient amounts of MCM2–7 before DNA replication (Figure).

The existence of the licensing checkpoint was proposed, whereby the cell senses a lack of chromatin-bound MCM2–7 and arrests the cell cycle in the G1 phase. In this review, we proposed a new hypothesis that cells do not sense the amount of MCM2–7 directly. Instead, the cells with reduced MCM2–7 expression have DNA damage during DNA replication in the previous cell cycle, and they arrest the cell cycle by detecting this DNA damage in the next G1 phase.

Figure: A schematic diagram of the assembling the MCM2–7 hexameric complex.

New assistant professor joins NIG as of April 1, 2023.

KAWAGUCHI,Akane : Kuraku Group • Molecular Life History Laboratory