Planetary period magnetic field oscillations in Saturn's magnetosphere: Postequinox abrupt nonmonotonic transitions to northern system dominance

[1] We examine the “planetary period” magnetic field oscillations observed in the “core” region of Saturn's magnetosphere (dipole L ≤ 12), on 56 near‐equatorial Cassini periapsis passes that took place between vernal equinox in August 2009 and November 2012. Previous studies have shown that these consist of the sum of two oscillations related to the northern and southern polar regions having differing amplitudes and periods that had reached near‐equal amplitudes and near‐converged periods ~10.68 h in the interval to ~1 year after equinox. The present analysis shows that an interval of strongly differing behavior then began ~1.5 years after equinox, in which abrupt changes in properties took place at ~6‐ to 8‐month intervals, with three clear transitions occurring in February 2011, August 2011, and April 2012, respectively. These are characterized by large simultaneous changes in the amplitudes of the two systems, together with small changes in period about otherwise near‐constant values of ~10.63 h for the northern system and ~10.69 h for the southern (thus, not reversed postequinox) and on occasion jumps in phase. The first transition produced a resumption of strong southern system dominance unexpected under northern spring conditions, while the second introduced comparably strong northern system dominance for the first time in these data. The third resulted in suppression of all core oscillations followed by re‐emergence of both systems on a time scale of ~85 days, with the northern system remaining dominant but not as strongly as before. This behavior poses interesting questions for presently proposed theoretical scenarios

Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing

<div><p>Zinc finger protein 809 (ZFP809) is a member of the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family, and is highly expressed in mouse immature cells. ZFP809 is known to inhibit the expression of transduced genes driven by Moloney murine leukemia virus (MoMLV)-typed retroviral vectors by binding to the primer binding site (PBS) located downstream of the MLV-long terminal repeat (LTR) of the vectors and recruiting protein complexes that introduce epigenetic silencing marks such as histone modifications and DNA methylation at the MLV-LTR. However, it remains undetermined what domains of ZFP809 among the KRAB domain at N-terminus and the seven zinc fingers are critical for gene silencing. In this study, we assessed subcellular localization, gene silencing ability, and binding ability to the PBS of a series of truncated and mutated ZFP809 proteins. We revealed the essential role of the KRAB A box for all functions assessed, together with the accessory roles of a subset of zinc fingers. Our data also suggest that interaction between KAP1 and the KRAB A box of ZFP809 is critical in KAP1-dependent control of gene silencing for ZFP809 targets.</p></div

Identification of ZFP809 sub-domains required for the PBS-dependent gene silencing of MLV-LTR driven transgene expression.

<p>(A) The structures of luciferase reporter vectors used are shown. Black and grey boxes at downstream of the CMV promoter represent the MLV- and dl587rev-derived PBS, respectively. The five nucleotide positions whose base sequence is different between two PBSs are underlined. 293FT cells were co-transfected with one of the reporter vectors and one of the pCMV/flag-X vectors (X denotes either of ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s001" target="_blank">S1B Fig</a>). RRR (relative response ratio) represents the value of the firefly luciferase activity relative to that of the <i>Renilla</i> luciferase activity (control) at 24 hours after transfection. Error bars show the standard deviation obtained from four independent experiments. Asterisks indicate statistical significance (<i>P</i> <0.05) with <i>Bonferroni</i> correction. (B) The structures of the retroviral GFP reporter vectors. When the RNA genome derived from retroviral vectors is reverse transcribed into an RNA-DNA duplex and then into double-stranded DNA, a part of 5' LTR is recombined to a part of 3' LTR. (C) 293FT cells transduced with one of the retroviral vectors (MLV/EGFP or MSCV/EGFP) were sorted based on EGFP expression, and transduced with one of the pLVSIN/CMV/flag-X/IRES/mCherry vectors or the control vector (pLVSIN/CMV/flag/IRES/mCherry). Then, the expression levels of EGFP and mCherry were analyzed at day 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s004" target="_blank">S4 Fig</a>) and day 15 (Fig 3C) after transduction.</p

Domain structures and subcellular localization of intact, truncated, and mutated ZFP809 proteins.

<p>(A) Schematic representation of the domain structures of the intact ZFP809 protein (1), a series of truncated proteins (2–10), and a mutated ZFP809 protein with three amino acid substitutions within the KRAB domain (11), assessed for their functionalities in this study. ZF and SD denote zinc finger and spacer domain, respectively. All constructs were attached with the FLAG tag at the N-terminus and cloned into the pLVSIN/IRES/mCherry vector (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s001" target="_blank">S1A Fig</a>), and referred to as pLVSIN/CMV/flag-X/ IRES/mCherry vectors (X denotes any of: intact ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB). ΔSD lacks a spacer domain of six amino acids at the C-terminus of ZFP809. (B) Confirmation of the size of the proteins expressed from the eleven vector constructs shown in Fig 1A. 293FT cells transduced with one of the eleven vector constructs were sorted based on mCherry expression. The sorted cells were subjected to Western blot analysis using anti-FLAG antibody (lane C, empty vector (pLVSIN/CMV/flag/IRES/mCherry); lanes 1 to 11, eleven vector constructs). The asterisk indicates non-specific bands. (C) Sub-nuclear localization of intact and truncated/mutated ZFP809 proteins exogenously expressed from lentiviral vectors in 293FT cells detected by immunostaining using the anit-FLAG antibody followed by confocal microscopic analysis. 293FT cells transduced with the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Anti-fibrillarin antibody was also used as a nucleolus marker. Two single-stained, merged, and DAPI-stained images are shown.</p

Assessment of truncated and mutated ZFP809 proteins for their binding ability to MLV-PBS.

<p>(A) EMSA of the MLV-PBS sequence with the series of intact/truncated/mutated ZFP809 proteins (8% gel). Asterisks indicate non-specific bands. (B) EMSA of the MLV-PBS sequence with the intact ZFP809 and ZF1-6 proteins in the presence of cold competitor DNA, anti-FLAG antibody, or anti-KAP1 antibody (8% gel). (C) and (D) EMSA of the MLV-PBS sequence with the ΔKRAB_A and mtKRAB proteins in the presence of cold competitor DNA, anti-FLAG antibody, or anti-KAP1 antibody electrophoresed on 8% gel (C) and 10% gel (D). Asterisks indicate non-specific bands. In “Control” lanes in A-D, the probe DNA was mixed with nuclear extracts from 293FT cells with lentiviral transduction of pLVSIN/CMV/flag/IRES/mCherry, and electrophoresed.</p

Co-localization and interaction of intact, truncated, and mutated ZFP809 proteins with KAP1.

<p>(A) 293FT cells transduced with pLVSINCMV/flag-X/IRES/mCherry vectors were immunostained with anti-FLAG antibody and anti-KAP1 antibody followed by confocal microscopic analysis. X denotes either of ZFP809, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔSD, ΔKRAB_A, or mtKRAB. 293FT cells with lentiviral transduction of the pLVSIN/CMV/flag/IRES/mCherry vector were used as the “Control”. Two single-stained, merged, and DAPI-stained images are shown. (B) Immunoprecipitation (IP) followed by Western blot analysis to assess the interaction of intact, truncated and mutated ZFP809 proteins with KAP1. Protein extracts from 293FT cells that were transduced with the lentiviral vectors corresponding to ZFP809, ΔZF, ΔKRAB_A, and mtKRAB (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.g001" target="_blank">Fig 1A</a>). The antibody used for IP and Western blotting is shown on the left and right sides of the band images, respectively. Anti-DDDDK tag antibody is equivalent to FLAG antibodies from Sigma. (C) A schematic model of the role of KAP1 in preventing the entry of ZFP809 into nucleoli. Our results shown in Fig 2A and B suggest that interaction of KAP1 with ZFP809 through its KRAB A box domain is necessary for the proper nuclear distribution of ZFP809 being excluded from nucleoli.</p

Models for ZFP809 sub-domains for nuclear localization, gene silencing and binding ability to MLV-PBS.

<p>ZFP809 contains a KRAB domain at the N-terminus and seven zinc fingers at the C-terminus. The nucleolar exclusion (under the nuclear localization) of ZFP809 is dependent of KAP1- KRAB_A domain interaction. Five (first to fifth) zinc fingers can bind to MLV-PBS in the absence of the KRAB_A domain (accordingly, without KAP1 interaction). However, the intact ZFP809 protein binds to MLV-PBS only when KAP1 interacts with the KRAB_A domain. Therefore, the interaction of KAP1 with the KRAB_A domain is essential for the PBS-dependent gene silencing primed by ZFP809.</p

KAP1 dependent biding ability of ZFP809 to MLV-PBS.

<p>(A) Confirmation of knockdown efficiency for KAP1. 293FT cells transduced with lentivirus from shRNA vectors for NC or KAP1 were sorted based on EGFP expression. The sorted cells were subjected to Western blot analysis using anti-KAP1 and Anti-β-Actin antibodies. (B) and (C) EMSA of the MLV-PBS sequence with the intact ZFP809 and ΔKRAB_A proteins in the presence of cold competitor DNA on an 8% gel (B) and a 10% gel (C). Asterisks indicate non-specific bands.</p

DNA Methyltransferase Inhibitor Zebularine Inhibits Human Hepatic Carcinoma Cells Proliferation and Induces Apoptosis

<div><p>Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-_D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both <em>in vitro</em> and <em>in vivo</em>. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21^WAF/CIP1 and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21^WAF/CIP1 and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.</p> </div

An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species

<div><p>Insulators are <i>cis</i>-elements that control the direction of enhancer and silencer activities (enhancer-blocking) and protect genes from silencing by heterochromatinization (barrier activity). Understanding insulators is critical to elucidate gene regulatory mechanisms at chromosomal domain levels. Here, we focused on a genomic region upstream of the mouse <i>Ccnb1ip1</i> (cyclin B1 interacting protein 1) gene that was methylated in E9.5 embryos of the C57BL/6 strain, but unmethylated in those of the 129X1/SvJ and JF1/Ms strains. We hypothesized the existence of an insulator-type element that prevents the spread of DNA methylation within the 1.8 kbp segment, and actually identified a 242-bp and a 185-bp fragments that were located adjacent to each other and showed insulator and enhancer activities, respectively, in reporter assays. We designated these genomic regions as the <i>Ccnb1ip1</i> insulator and the <i>Ccnb1ip1</i> enhancer. The <i>Ccnb1ip1</i> insulator showed enhancer-blocking activity in the luciferase assays and barrier activity in the colony formation assays. Further examination of the <i>Ccnb1ip1</i> locus in other mammalian species revealed that the insulator and enhancer are highly conserved among a wide variety of species, and are located immediately upstream of the transcriptional start site of <i>Ccnb1ip1</i>. These newly identified cis-elements may be involved in transcriptional regulation of <i>Ccnb1ip1</i>, which is important in meiotic crossing-over and G2/M transition of the mitotic cell cycle.</p></div