Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the singlecell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.coupled oscillators | oscillations | circadian rhythms | gating

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Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP.

SummaryWe identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3Sci), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3Sci/+ SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3Sci/+ SCN slices. In conclusion, by cloning Zfhx3Sci, we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

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Novel dual thermoresponsive block copolymers displaying lower critical solution temperature (LCST) and upper critical solution temperature (UCST) were synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization of two proline-based monomers. Poly(N-acryloyl-l-proline methyl ester), poly(A-Pro-OMe), was selected as a thermoresponsive segment, whereas poly(N-acryloyl-4-trans-hydroxy-l-proline), poly(A-Hyp-OH), could be regarded as a water-soluble polymer. The block copolymer having suitable comonomer composition (A-Pro-OMe/A-Hyp-OH = 27/73) exhibited soluble−insoluble−soluble transition with lower (LCST = 19−21 °C) and upper (UCST = 39−45 °C) critical solution temperatures in acidic water. The comonomer composition of poly(A-Pro-OMe)-b-poly(A-Hyp-OH) and pH value in the aqueous solution were found to affect characteristic thermoresponsive behaviors. The temperature-dependent assembled structures and chiroptical properties were evaluated by dynamic light scattering (DLS) and circular dichroism (CD) measurements. Another type of dual thermosensitive block copolymers with blocks having two different LCSTs, poly(A-Pro-OMe)-b-poly(A-Hyp-OMe), were prepared by the methylation of the carboxylic acid groups in poly(A-Pro-OMe)-b-poly(A-Hyp-OH), and their temperature-dependent solution behaviors were investigated. To the best of our knowledge, this is the first report of the dual thermoresponsive system, which can be changed from a system exhibiting LCST and UCST into another one having two different LCSTs by a simple methylation reaction.

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The SET-CAN fusion gene is the product of a chromosomal rearrangement found on 9q34 associated with an acute undifferentiated leukemia. SET-CAN encodes an almost complete SET protein fused to the C-terminal two-thirds of CAN. SET is also known as TAF-I␤, a histone chaperone and intracellular inhibitor of protein phosphatase 2A, whereas CAN is identical to Nup214, a nucleoporin protein. To obtain insight into the leukemogenic function of SET/TAF-I␤-CAN/ Nup214, we have examined its subcellular localization. Immunofluorescence analyses showed that SET/TAF-I␤ and CAN/Nup214 are found in the nucleus and the nuclear envelope, respectively, whereas the majority of SET/TAF-I␤-CAN/Nup214 is localized in the nucleus. SET/TAF-I␤-CAN/ Nup214 interacted with hCRM1, one of the nuclear export factors, and caused aberrant intracellular localization of hCRM1. In cells expressing SET/TAF-I␤-CAN/Nup214, a protein containing a nuclear export signal accumulated in the nucleus. The export of this protein was partially restored by overexpression of hCRM1. These results suggest that aberrantly localized molecules associated with SET/TAF-I␤-CAN/ Nup214 may be involved in oncogenesis.