Figure S1. Full plasmid sequences of the Cas9, gRNA, and donor vectors. Green, orange, blue, red,... more Figure S1. Full plasmid sequences of the Cas9, gRNA, and donor vectors. Green, orange, blue, red, light blue, gray letters indicate the promoter, coding sequence, terminator, gRNA, homologous region, and bar gene cassette, respectively. 20-bp target sequences in gRNA are underlined.
All teratomas from miPSCs or miPSCs-p27 injected in nude mice. Teratomas from miPSCs and miPSCs-p... more All teratomas from miPSCs or miPSCs-p27 injected in nude mice. Teratomas from miPSCs and miPSCs-p27 injected mice are shown (Aâ D) and (Eâ H), respectively. (PDF 828 kb)
Expression profile of stem cell factors in miPSCs-p27. RT-PCR analysis of stem cell marker genes ... more Expression profile of stem cell factors in miPSCs-p27. RT-PCR analysis of stem cell marker genes (A) and relative intensities (B) of miPSCs and miPSCs-p27 are shown. Error bars correspond to the SEM (n = 3). *P
Effects of p27 overexpression on EB formation. Percentages of wells which have EBs and beating EB... more Effects of p27 overexpression on EB formation. Percentages of wells which have EBs and beating EBs are shown in A and B, respectively. Error bars correspond to the SEM (n = 3). *P
Purification of p27 expressing miPSCs. Flow cytometric analysis of miPSCs (A), or p27-2A-mRFP sta... more Purification of p27 expressing miPSCs. Flow cytometric analysis of miPSCs (A), or p27-2A-mRFP stable transfected miPSCs (miPSCs-p27) before (B) and after (C) purification by cell sorter. Intensities of mRFP fluorescent were plotted against side scatters (SSC). The area surrounded by red line in B was sorted. (PDF 348 kb)
Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the compos... more Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed molecular mechanisms regulating circadian physiology of the intestine remain largely unknown. The lack of appropriate human model systems that enable organ- and/or diseasespecific interrogation of clock functions is a major obstacle hindering advancements of translational applications using chronotherapy. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids (HIEs) possess robust circadian rhythms, and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses. RNA-Seq data show ~4% of genes are rhythmically expressed in HIEs. Remarkably, we obs...
Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidire... more Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidirectional communication, resulting in circadian clock-gated cell division cycles in species ranging from cyanobacteria to mammals. The identified mechanisms for this phenomenon have expanded beyond intracellular molecular coupling components to include intercellular connections. However, detailed molecular mechanisms, dynamics, and physiological functions of the circadian clock and cell cycle as coupled oscillators remain largely unknown. In this review, we discuss current understanding of this connection in light of recent findings that have uncovered intercellular coupling between the circadian clock in Paneth cells and the cell cycle in intestinal stem cells via WNT signaling. This extends the impact of circadian rhythms regulating the timing of cell divisions beyond the intracellular domain of homogenous cell populations into dynamic, multicellular systems. In-depth understanding of th...
Although the coupling between circadian and cell cycles allows circadian clocks to gate cell divi... more Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify D...
Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intrace... more Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.
Figure S1. Full plasmid sequences of the Cas9, gRNA, and donor vectors. Green, orange, blue, red,... more Figure S1. Full plasmid sequences of the Cas9, gRNA, and donor vectors. Green, orange, blue, red, light blue, gray letters indicate the promoter, coding sequence, terminator, gRNA, homologous region, and bar gene cassette, respectively. 20-bp target sequences in gRNA are underlined.
All teratomas from miPSCs or miPSCs-p27 injected in nude mice. Teratomas from miPSCs and miPSCs-p... more All teratomas from miPSCs or miPSCs-p27 injected in nude mice. Teratomas from miPSCs and miPSCs-p27 injected mice are shown (Aâ D) and (Eâ H), respectively. (PDF 828 kb)
Expression profile of stem cell factors in miPSCs-p27. RT-PCR analysis of stem cell marker genes ... more Expression profile of stem cell factors in miPSCs-p27. RT-PCR analysis of stem cell marker genes (A) and relative intensities (B) of miPSCs and miPSCs-p27 are shown. Error bars correspond to the SEM (n = 3). *P
Effects of p27 overexpression on EB formation. Percentages of wells which have EBs and beating EB... more Effects of p27 overexpression on EB formation. Percentages of wells which have EBs and beating EBs are shown in A and B, respectively. Error bars correspond to the SEM (n = 3). *P
Purification of p27 expressing miPSCs. Flow cytometric analysis of miPSCs (A), or p27-2A-mRFP sta... more Purification of p27 expressing miPSCs. Flow cytometric analysis of miPSCs (A), or p27-2A-mRFP stable transfected miPSCs (miPSCs-p27) before (B) and after (C) purification by cell sorter. Intensities of mRFP fluorescent were plotted against side scatters (SSC). The area surrounded by red line in B was sorted. (PDF 348 kb)
Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the compos... more Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed molecular mechanisms regulating circadian physiology of the intestine remain largely unknown. The lack of appropriate human model systems that enable organ- and/or diseasespecific interrogation of clock functions is a major obstacle hindering advancements of translational applications using chronotherapy. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids (HIEs) possess robust circadian rhythms, and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses. RNA-Seq data show ~4% of genes are rhythmically expressed in HIEs. Remarkably, we obs...
Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidire... more Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidirectional communication, resulting in circadian clock-gated cell division cycles in species ranging from cyanobacteria to mammals. The identified mechanisms for this phenomenon have expanded beyond intracellular molecular coupling components to include intercellular connections. However, detailed molecular mechanisms, dynamics, and physiological functions of the circadian clock and cell cycle as coupled oscillators remain largely unknown. In this review, we discuss current understanding of this connection in light of recent findings that have uncovered intercellular coupling between the circadian clock in Paneth cells and the cell cycle in intestinal stem cells via WNT signaling. This extends the impact of circadian rhythms regulating the timing of cell divisions beyond the intracellular domain of homogenous cell populations into dynamic, multicellular systems. In-depth understanding of th...
Although the coupling between circadian and cell cycles allows circadian clocks to gate cell divi... more Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify D...
Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intrace... more Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.
Uploads
Papers by Toru Matsu-ura