ALBERT

Description: Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.

Description: Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

Description: Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (~30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.

Description: Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata , whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this ‘internal standard’ calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

Description: In 2010, a wide-angle seismic reflection/refraction profile was acquired along the Hecataeus Rise, an area of shallow seabed immediately south of Cyprus in the eastern Mediterranean. The profile crossed from the Hecataeus Rise, through the Cyprus Arc to the Levantine Basin beyond. Due to the short length of the profile and the corresponding lack of deep ray coverage, velocity modelling was complemented by gravity modelling to gain constraints on deep crustal structure. The resultant model reveals velocities for the Hecataeus Rise that show no evidence of shallow ophiolites like those seen on mainland Cyprus, and the velocities are not diagnostic of a unique crustal affinity. Low-velocity sediments make up at least 7 km of the upper structure of Hecataeus Rise and these sediments overlie a two-layered crust. From the gravity modelling, the combined sediments and crust of Hecataeus Rise appear to be thinner than the Eratosthenes Seamount block to the southwest. A high-velocity lower crustal block is modelled under the seaward edge of Hecataeus Rise and, based on the gravity modelling, is inferred to extend landwards beneath the Rise. Similar high-velocity blocks were identified on the southwestern edge of Hecataeus Rise along nearby refraction lines and were interpreted as remnant Tethyan oceanic crust, foundered in the Cyprus Arc, along which subduction has ceased in this area. Given the thin two-layered crust beneath a thick accumulation of sediments modelled for Hecataeus Rise, we interpret that Hecataeus Rise represents a collage of oceanic fragments, accreted together within the failed subduction zone. Outboard of the crust of Hecataeus Rise, a 5-km deep low-velocity basin, possibly an accretionary wedge, is imaged that appears to correspond with the Cyprus Arc deformation zone imaged on both coincident and along-strike seismic reflection lines. A similar and wider feature is observed on seismic refraction lines to the west and combined, these may be revealing an eastward tapering zone of crustal deformation. To the south of the profile, the Levantine Basin appears undisturbed by the collision to the north and exhibits a uniform and homogeneous velocity structure.

Description: This study investigates pressure effects on the magnetic properties of non-interacting single-domain (SD) magnetite. Using a high-pressure cell specially designed for a Magnetic Property Measurement System, magnetic hysteresis measurements were conducted under high pressures of up to 1 GPa on natural plagioclase crystals containing much acicular SD magnetite. Coercivity and saturation magnetization were nearly constant with pressure, while saturation remanent magnetization and coercivity of remanence decreased with pressure at moderate rates of –8 per cent GPa –1 and –18 per cent GPa –1 , respectively. These results suggest that temperature effects govern the magnetic behaviour of acicular SD magnetite grains in the middle and lower crusts.

Description: We studied ionospheric responses to the 2012 April 11 M w 8.6 North Sumatra earthquake using total electron content (TEC) measurements with the regional Global Navigation Satellite System network. This earthquake ruptured the oceanic lithosphere off the Indian Ocean coast of North Sumatra, and is known as the largest strike-slip earthquake ever recorded. Coseismic ionospheric disturbances (CIDs) with rapid TEC enhancement of a few TEC units propagated northward with a speed of acoustic waves (~1 km s –1 ). Resonant atmospheric oscillation with a frequency ~4 mHz have been found as monochromatic oscillation of TEC lasting for an hour after the main shock and the largest aftershock. We compared CID amplitudes of 21 earthquakes world-wide with moment magnitudes ( M w ) 6.6–9.2. They roughly obeyed a law such that CID amplitude increases by two orders of magnitude for the M w increase of three. The 2012 North Sumatra earthquakes slightly deviated negatively from the trend possibly reflecting their strike-slip mechanisms, that is small vertical crustal movements for their magnitudes.

Description: Data on the evolution of Earth's magnetic field intensity are important for understanding the geodynamo and planetary evolution. However, the paleomagnetic record in rocks may be adversely affected by many physical processes, which must be taken into account when analysing the palaeointensity database. This is especially important in the light of an ongoing debate regarding core thermal conductivity values, and how these relate to the Precambrian geodynamo. Here, we demonstrate that several data sets in the Precambrian palaeointensity database overestimate the true paleofield strength due to the presence of non-ideal carriers of palaeointensity signals and/or viscous re-magnetizations. When the palaeointensity overestimates are removed, the Precambrian database does not indicate a robust change in geomagnetic field intensity during the Mesoproterozoic. These findings call into question the recent claim that the solid inner core formed in the Mesoproterozoic, hence constraining the thermal conductivity in the core to ‘moderate’ values. Instead, our analyses indicate that the presently available palaeointensity data are insufficient in number and quality to constrain the timing of solid inner core formation, or the outstanding problem of core thermal conductivity. Very young or very old inner core ages (and attendant high or low core thermal conductivity values) are consistent with the presently known history of Earth's field strength. More promising available data sets that reflect long-term core structure are geomagnetic reversal rate and field morphology. The latter suggests changes that may reflect differences in Archean to Proterozoic core stratification, whereas the former suggest an interval of geodynamo hyperactivity at ca. 550 Ma.

Description: Was the Yukon-Tanana Terrane (YTT), a California-sized part of south-central Yukon, an autochthonous or para-autochthonous part of northern British Columbia in the Early Cretaceous or was it part of a proposed allochthonous ‘Baja B.C.’ continent offshore of southern California? To answer this fundamental question, a paleomagnetic study has been completed on 347 specimens from 24 sites in the 114.7 ± 1.1 Ma Quiet Lake batholith. This 1300 km 2 pluton is composed mostly of massive medium-to-coarse grained biotite quartz monzonite that exhibits no evidence of either deformation or metamorphism, and that intrudes metamorphosed pre-Cretaceous basement rocks of the YTT in southern Yukon. The paleomagnetic analysis utilized thermal and alternating field step demagnetization, and saturation isothermal remanence methods. A well-defined characteristic remanent magnetization (ChRM) direction was isolated throughout the 500–585 °C temperature range at Decl. = 340.6°, Incl. = 77.4° ( N = 14 sites, k = 51.2, A 95 = 5.6°). The ChRM resides in magnetite with a low titanium content and is interpreted to be a primary thermoremanent magnetization. After correction for 490 km of geologically demonstrable dextral displacement on the inboard Tintina fault zone, the Quiet Lake batholith's paleopole is not significantly different at 95 per cent confidence from the co-eval 115 Ma reference paleopole for North America, giving non-significant translation and rotation estimates of 1.4° ± 5.1° (1) northwestwards and 10° ± 13° (1) clockwise, respectively. Thus, this is the first Early Cretaceous paleopole to show clearly that the YTT in Yukon is a para-autochthon that was part of North America's continental margin at that time. Further, after correction for Tintina fault displacement, the eight available Mesozoic YTT paleopoles agree closely with the North American apparent polar wander path (APWP). In contrast, the 22 paleopoles from the Intermontane Belt show the expected behaviour of an allochthonous thin-skin of terranes (i.e. ‘Baja B.C.’ behaviour) and record ~8° of northward translation and ~50° of clockwise rotation relative to both the YTT and North American APWPs during the Mesozoic and Paleogene.

Description: Understanding the contribution of biogenic magnetic particles into sedimentary assemblages is a current challenge in palaeomagnetism. It has been demonstrated recently that magnetic particles produced through biologically controlled mineralization processes, such as magnetosomes from magnetotactic bacteria, contribute to the recording of natural remanent magnetization in marine and lacustrian sediments. Contributions from other, biologically induced, mineralization types, which are known from multiple laboratory experiments to include magnetic minerals, remain largely unknown. Here, we report magnetic properties of iron minerals formed by a community of iron- and manganese-reducing bacteria isolated from a natural groundwater deposit during a 2 yr long incubation experiment. The main iron phases of the biomineralized mass are lepidocrocite, goethite and magnetite, each of which has environmental significance. Unlike the majority of the previous studies that reported superparamagnetic grain size, and thus no remanence carrying capacity of biologically induced magnetite, hysteresis and first-order reversal curves measurements in our study have not detected significant superparamagnetic contribution. The biomineralized mass, instead, contains a mixture of single-domain to pseudo-single-domain and multidomain magnetite particles that are capable of carrying a stable chemical remanent magnetization. Isothermal remanent magnetization acquisition parameters and first-order reversal curves signatures of the biomineralized samples deviate from previously proposed criteria for the distinction of extracellular (biologically induced) magnetic particles in mixtures. Given its potential significance as a carrier of natural remanent magnetization, environmental requirements, distribution in nature and the efficiency in the geomagnetic field recording by biologically induced mineralization need comprehensive investigation.