Biology subjects

Murayama, Y.

Publications and source records attributed to Murayama, Y..

13 recordsLinked to original sources

bioRxiv2025

FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

Amino acid catabolism during fasting requires coordinated nitrogen disposal and glucose production, but the transcriptional logic linking the urea cycle to gluconeogenesis remains unclear. Forkhead box O (FoxO) transcription factors are key regulators of fasting metabolism, yet their role in controlling urea cycle genes has not been fully defined. Here we identify FoxOs as direct regulators of hepatic argininosuccinate synthase 1 (Ass1). Because FoxOs often act through Kruppel-like factor 15 (Klf15) in amino acid metabolism, we tested whether Ass1 regulation requires Klf15. Acute hepatic FoxO1/3a knockdown in fasted mice selectively reduced Ass1 expression, lowered blood glucose, and shifted urea-cycle amino acids, with arginine decreased and ornithine increased, even in Klf15-deficient livers. Silencing Ass1 phenocopied these metabolic effects, indicating that Ass1 mediates a key FoxO-dependent branch of fasting adaptation. Mechanistically, we mapped a functional FoxO-binding element within an upstream Ass1 enhancer: FoxO1/3a activated the enhancer in reporter assays, EMSA confirmed binding, and in vivo luciferase imaging and liver ChIP demonstrated fasting-inducible enhancer activity and FoxO occupancy. Collectively, these findings establish a FoxO-Ass1 axis that couples ureagenesis to gluconeogenesis and supports metabolic flexibility during fasting. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/695746v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@94ff36org.highwire.dtl.DTLVardef@1ff4f3forg.highwire.dtl.DTLVardef@abd0e5org.highwire.dtl.DTLVardef@1f8d9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

ATAC-seq of Low-Input and Cryopreserved Primordial Germ Cells Reveals Functional Enhancers

Cell fate specification during development is orchestrated by dynamic changes in gene expression, driven by the accessibility of cis-regulatory elements. To profile such functional regulatory elements at the genome-wide level, bulk ATAC-seq is a powerful method. Yet, its application has still been technically limited in rare cell types. Here, we optimized and titrated the ATAC-seq protocol for cultured chicken primordial germ cells (PGCs) and obtained high-quality chromatin accessibility profiles from as few as 200 cells, even after cryopreservation. Integrative analysis of our ATAC-seq with published RNA-seq and tissue ATAC-seq datasets identified over 10,000 PGC-specific accessible chromatin regions (ACRs), many absent from somatic tissues. Reporter assays demonstrated activity of selected enhancers in cultured PGCs, while transcriptome integration revealed preferential association of PGC-specific ACRs with genes highly expressed at embryonic day 2.5. In vivo transplantation confirmed that these enhancers are active in an early-stage-specific manner, but become silenced upon gonadal settlement. In conclusion, this streamlined method provides a practical and cost-effective approach to the epigenomic analysis of scarce cell populations, facilitating future investigations in developmental biology, germline research, and beyond.

developmental biology↗

bioRxiv2025

Submolecular video-imaging of the Smc5/6 complex topologically bound to DNA

Structural maintenance of chromosomes (SMC) complexes are ring-shaped ATPases that hold distinct DNA segments together to regulate various chromosome structures and functions. However, their dynamic mechanisms of DNA binding and processing remain poorly understood. Here, using high-speed atomic force microscopy, we directly visualized the dynamics of the Smc5/6 complex on DNA at submolecular resolution, resolving its individual domains. ATP-bound Smc5/6 stably binds DNA via the ATPase head domain, whereas following ATP hydrolysis, the DNA is entrapped within the SMC compartment and positioned around the opposite hinge domain. Furthermore, Smc5/6 topologically entraps two DNA segments together and stabilizes the DNA twist structures, promoting DNA compaction. Our findings provide a visual demonstration of how an SMC complex employs its ring architecture to facilitate distinct DNA binding modes.

biophysics↗

bioRxiv2024

Minimal protein list for chromatin replication-coupled symmetric histone recycling revealed through in vitro reconstitution

Upon eukaryotic DNA replication, symmetric histone recycling from parental to daughter strands is vital for transmitting epigenetic information to the next generations. Recent genome-wide sequencing studies have identified several protein regions contributing to symmetric histone recycling in a cell. However, a minimal list of proteins for symmetric histone recycling remains unknown. Here, we successfully reconstituted histone recycling with [~]30 purified proteins and analyzed the products digested by Micrococcal nuclease with the newly developed pipeline called a Repli-pore-seq in which nanopore sequencing and deep-learning-based classification were combined. As a result, we confirmed that the histones were recycled symmetrically to the lagging and leading strands. The recycled histones form tetrasomes or hexasomes and are deposited on the DNA sequences energetically favorable for forming nucleosomes. We also observed the discordance of the recycled position between lagging and leading strands on the GC-rich DNA sequences. Among proteins dispensable for chromatin replication, the lagging-strand maturation factors, Fen1/Cdc9, were also dispensable for symmetric recycling. The removal of Pol {delta} disrupted the recycling symmetry, and that of Ctf4 or Csm3/Tof1 altered the recycling position. These findings provide critical insights into the molecular players and mechanisms underlying symmetric histone recycling.

biophysics↗

bioRxiv2024

Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex

The 5-3 exoribonuclease Rat1 (or Xrn2) plays a pivotal role in termination of mRNA transcription by eukaryotic RNA polymerase II (RNAPII). Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a "torpedo" to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of a Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast Komagataella phaffii. The Rat1-Rai1-Rtt103 complex structure revealed that Rat1 and Rai1 form a heterotetramer, with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and directly binds to the RNA exit site of transcribing RNAPII to extract RNA into the Rat1 nuclease active site. This interaction changes the RNA path in favor of termination (the "pre-termination" state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the "post-termination" states. These structures shed light on the mechanism of eukaryotic mRNA transcription termination.

biochemistry↗

bioRxiv2024

Structural insight into bacterial co-transcriptional translation initiation

In bacteria, transcription and translation are tightly coupled, forming a transcription-translation complex (TTC) between RNA polymerase (RNAP) and the ribosome. As nascent mRNA emerging from RNAP is susceptible to ribonuclease digestion, undesired RNA folding, and R-loop formation, immediate TTC formation is important. Here, we report the cryo-electron microscopy structures that capture the translation initiation complex assembly on transcribing RNAP. As a short mRNA emerging from RNAP, the 30S ribosomal subunit binds the RNAP on its inter-subunit side, interacting with the mRNA 5-region and the initiator tRNA. The RNAP could relocate to the canonical mRNA-entry site of 30S, threading the mRNA into a path formed between RNAP and 30S. The subsequent 50S joining establishes a TTC. These structures illustrate the transcription-coupled translation initiation, while protecting mRNA. One-Sentence SummaryCryo-EM captures the ribosome assembly on transcribing RNA polymerase in the presence of translation initiation factors.

biochemistry↗

bioRxiv2024

Molecular mechanism of parental H3/H4 recycling at a replication fork

In eukaryotic chromatin replication, faithful recycling of histones from parental DNA to replicated leading and lagging strands is essential for maintaining epigenetic information across generations. A recent experimental study has revealed that disrupting interactions between the N-terminal disordered tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer, a carrier of epigenetic modifications, perturbs their faithful recycling. However, the molecular pathways via which the H3/H4 tetramer attached to Mcm2 is recycled to the replicated strands have yet to be deciphered. Furthermore, the factors that regulate the ratio recycled to each strand and the destination location still need to be discovered. The elucidation requires visualization of a structural trajectory from H3/H4 bound to Mcm2 until recycled to the replicated strands. In this study, we performed molecular dynamics simulations of yeast DNA replication machinery (Mcm2-7, Cdc45, GINS, Pol {varepsilon}, and RPA), an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways even without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding to the ssDNA portion of the lagging strand regulated the ratio recycled to each strand, whereas DNA bending by Pol {varepsilon} modulated the destination location. Together, the simulations in this study provided testable hypotheses, which are vital for clarifying intracellular histone recycling controlled by the cooperation of many histone chaperones.

biophysics↗

bioRxiv2023

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrate increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

bioengineering↗

bioRxiv2022

Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the ATP-dependent RNA translocase/helicase Rho, which causes the dissociation of RNA/DNA from RNAP elongation complex (EC). However, structural basis of the interplay between Rho and RNAP remains obscure. Here we report the cryo-electron microscopy structure of the Rho-engaged EC. The Rho hexamer binds RNAP through the C-terminal domains, which surround the RNA-exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to the Rho central channel. The {beta}-flap tip at the RNA exit is critical to the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho-binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation. TeaserCryo-EM captures the structure of an RNA polymerase elongation complex engaged with the termination factor Rho.

biochemistry↗

bioRxiv2021

Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

The ER-embedded transcription factors, sterol-regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs), but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/457590v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@591eb2org.highwire.dtl.DTLVardef@ee6361org.highwire.dtl.DTLVardef@be893eorg.highwire.dtl.DTLVardef@1247d38_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Enterohepatic transcription factor CREB3L3 protects atherosclerosis via SREBP competitive inhibition

CREB3L3 is a membrane-bound transcription factor to maintain lipid metabolism in the liver and small intestine. CREB3L3 ablation in Ldlr-/- mice exacerbated hyperlipidemia with remnant ApoB-containing lipoprotein accumulation, developing enhanced aortic atheroma formation, whose extent was additive between liver- and intestine-specific deletion. Conversely, hepatic nuclear CREB3L3 overexpression markedly suppressed atherosclerosis with amelioration of hyperlipidemia. CREB3L3 directly upregulates anti-atherogenic FGF21 and ApoA4, whereas antagonizes hepatic SREBP-mediated lipogenic and cholesterogenic genes and regulates LXR-regulated genes involved in intestinal transport of cholesterol. CREB3L3 deficiency accumulates nuclear SREBP proteins. Because both transcriptional factors share the cleavage system for nuclear transactivation, full-length CREB3L3 and SREBPs on endoplasmic reticulum (ER) functionally inhibit each other. CREB3L3 competitively antagonizes SREBPs for ER-Golgi transport, resulting in ER retention and proteolytic activation inhibition at Golgi, and vice versa. Collectively, due to this new mechanistic interaction between CREB3L3 and SREBPs under atherogenic conditions, CREB3L3 has multi-potent protective effects against atherosclerosis.

physiology↗

bioRxiv2019

Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

molecular biology↗

bioRxiv2019

Rad51 Interaction Analysis Reveals a Functional Interplay Among Recombination Auxiliary Factors

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

molecular biology↗