Biology subjects

Murakawa, Y.

Publications and source records attributed to Murakawa, Y..

9 recordsLinked to original sources

bioRxiv2025

Cellular fitness is determined by ribosomal protein S12-mediated release of a truncated Xrp1

Multicellular tissues require continuous optimization to maintain their structure and function by actively eliminating viable, yet unfit cells via a mechanism known as cell competition. During this process, unfit loser cells commonly upregulate the C/EBP family transcription factor Xrp1, which causes their elimination, indicating that intracellular Xrp1 levels determine cellular fitness. However, despite its central role, the mechanism by which Xrp1 is upregulated in unfit cells remains unknown. Here, we show that Xrp1 is upregulated through a previously unrecognized post-transcriptional regulatory mechanism mediated by the ribosomal protein S12 (RpS12). Surprisingly, Xrp1 mRNA is abundantly expressed even in wild-type cells, yet it is not translated. This is due to an upstream open reading frame (uORF) located in the 5UTR of the initial coding exon of Xrp1 mRNA, which inhibits the translation of the main Xrp1 ORF. Intriguingly, in unfit cells, RpS12 causes splicing-mediated skipping of this initial exon, leading to the use of an alternate start codon that generates a short isoform of Xrp1 protein, causing cell death. Notably, protein structural analysis reveals that RpS12 is highly homologous to that of a spliceosomal component SNU13, suggesting the role of RpS12 in directly regulating alternative splicing of Xrp1 mRNA. Our findings thus provide not only a mechanistic insights into how cellular fitness is determined but also how tissue optimization is achieved.

cell biology↗

bioRxiv2024

Predicted constrained accessible regions mark regulatory elements and causal variants

Open chromatin regions (OCRs) define cell-type-specific regulatory elements across the genome, yet their functional significance varies, making it challenging to pinpoint biologically essential regions. Here, we introduce CAMBUS (Chromatin Accessibility Mutation Burden Score), a machine-learning framework that identifies active and evolutionarily constrained OCRs by leveraging surrounding DNA sequences. Applying CAMBUS to 29 immune cell types, we identified 66,043 constrained OCRs, which were substantially enriched in the known constraint genome (odds ratio=11.45 (95% confidence interval 9.33-14.05), P=4.7 x 10-68), while 90% of these OCRs were not prioritized by existing constraint metrics. These OCRs were highly enriched for known enhancers and super-enhancers, independent of known epigenetic markers and annotated regions, and overlapped with regulatory elements implicated in immune-mediated diseases and experimentally validated functional variants, including rare variants, particularly in leukocyte-related traits. Furthermore, CAMBUS revealed cell-type-specific transcriptional regulatory landscapes, linking genetic constraint with gene regulation in immune cells and identifying plausible connections between 1,533 causal variants and 70 complex traits. By defining biologically constrained regulatory elements at high resolution, CAMBUS provides a framework for understanding the selective pressures shaping the non-coding genome and its role in human health and disease.

genetics↗

bioRxiv2024

Atlas of Cynomolgus Macaque Hematopoiesis

Self-renewal and differentiation are inherent properties of hematopoietic stem cells (HSCs) that are necessary to support hematopoiesis; however, the underlying mechanisms, especially in human, remain unclear. Here, using the cynomolgus macaque as a surrogate model, we develop a new gating strategy to isolate with high purity transplantable cynomolgus HSCs and generated a single-cell transcriptomic map of cynomolgus HSCs and progenitor cells -- that covering gestational periods previously not analyzed in human. We show that hematopoietic cells from the late-1st to early-3rd trimester fetal liver and late-2nd trimester and thereafter bone marrow have repopulating potential, closely mimicking humans. Unexpectedly however, we found unlike in human, cynomolgus HSCs express CD38 but not CD33, indicating that these cellular counterparts are molecularly distinct. Our transcriptomic analysis reveals the presence of a direct differentiation pathway from HSCs to megakaryocyte lineages, lineage-primed multipotent progenitors and also identified putative HSC surface markers. Taken together, our comprehensive dataset highlights not only the utility of cynomolgus monkeys as model systems to study hematopoiesis but also their potential for translational applications.

cell biology↗

bioRxiv2022

Mobile elements in human population-specific genome and phenotype divergence

Mobile genetic elements (MEs) are heritable mutagens that contribute to divergence between lineages by recursively generating structural variants. ME variants (MEVs) are difficult to genotype, obscuring their impact on recent genome and trait diversification. We developed a tool that uses short-read sequence data to accurately genotype MEVs, enabling us to study them using statistical genetics methods in global human genomes. We observe population-specific differences in the distribution of Alu insertions that distinguish Japanese from other populations. We integrated MEVs with epigenomic and expression quantitative trait loci (eQTL) maps to determine how they impact traits. This reveals coherent patterns by which specific MEs regulate tissue-specific gene expression, including creating or attenuating enhancers and recruiting post-transcriptional regulators. We pinpoint MEVs as genetic causes of disease risk, including a LINE-1 insertion linked to keloid and other diseases of fibroblast inflammation, by introducing MEVs into the genome-wide association study (GWAS) framework. In addition to nominating previously-hidden MEVs as causes of human diseases, this work highlights MEs as accelerators of human population divergence and begins to decipher the semantics of MEs.

genetics↗

bioRxiv2020

Chromosomal-scale De novo Genome Assemblies of Cynomolgus Macaque and Common Marmoset

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Their genomes were sequenced and assembled initially using short-read sequences, with the advent of massively parallel sequencing. However, the resulting contig sequences tended to remain fragmentary, and long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. Firstly we assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data. The scaffolded sequences obtained were further refined based on assembly results of alternate de novo assemblies and Hi-C contact maps by resolving identified inconsistencies. The final assemblies achieved N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively, and the numbers of scaffolds longer than 10Mb are equal to their chromosome numbers. The high fidelity of our assembly is ascertained by concordance to the BAC-end read pairs observed for common marmoset, as well as a high resemblance of their karyotypic organization. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.

genomics↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗

bioRxiv2019

Dynamics and Topology of Human Transcribed Cis-regulatory Elements

Promoters and enhancers are key cis-regulatory elements, but how they orchestrate to generate cell-type-specific transcriptomes remains elusive. We developed a simple and robust approach to globally determine 5-ends of nascent RNAs (NET-CAGE) in diverse cells and tissues, thereby sensitively detecting unstable transcripts including enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, uncovering the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without influence of RNA turnover, and identified enhancer-promoter pairs which were simultaneously activated upon cellular stimulation. By integrating NET-CAGE data with chromatin interaction maps, we revealed that cis-regulatory elements are topologically connected according to their cell-type specificity. We discovered new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our collection of NET-CAGE data from human and mouse significantly expanded the FANTOM5 catalogue of transcribed enhancers, with broad applicability to biomedical research. (148 words)

genomics↗

bioRxiv2019

Codon Optimality Confers GC-Rich-Induced Stability to mRNAs in Humans

Codon bias has been implicated as one of the major factors contributing to mRNA stability in yeast. However, the effects of codon-bias on mRNA stability remain unclear in humans. Here we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias different from that of yeast. Bioinformatics analysis showed that codons could be clustered into two distinct groups - codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3); the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3 content entails proportionately higher GC content. Through bioinformatics, ribosome profiling and in vitro analysis, we show that decoupling of the effects of codon bias reveals two modes of mRNA regulation, GC3- and GC-content dependent. Employing an immunoprecipitation-based strategy, we identified ILF2 as an RNA binding protein that differentially regulates global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.

molecular biology↗

bioRxiv2018

Selective activation of alternative MYC core promoters by Wnt-responsive enhancers

In Metazoans, transcription of most genes is driven through the use of multiple alternative promoters. Although the precise spatio-temporal regulation of alternative promoters is important for proper gene expression, the mechanism that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types, and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in the colon carcinoma cell line HCT-116, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using both transgenic reporter assays and in the context of the endogenous Myc locus upon Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by distinct core promoter elements present in the two MYC promoters. Taken together, our results provide new insights into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more diverse combinatorial regulation of transcription initiation.

genetics↗