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.