Site-Selective Monitoring of the Interaction of the SRA Domain of UHRF1 with Target DNA Sequences Labeled with 2‑Aminopurine

UHRF1 plays a central role in the maintenance and transmission of epigenetic modifications by recruiting DNMT1 to hemimethylated CpG sites via its SET and RING-associated (SRA) domain, ensuring error-free duplication of methylation profiles. To characterize SRA-induced changes in the conformation and dynamics of a target 12 bp DNA duplex as a function of the methylation status, we labeled duplexes by the environment-sensitive probe 2-aminopurine (2-Ap) at various positions near or far from the central CpG recognition site containing either a nonmodified cytosine (NM duplex), a methylated cytosine (HM duplex), or methylated cytosines on both strands (BM duplex). Steady-state and time-resolved fluorescence indicated that binding of SRA induced modest conformational and dynamical changes in NM, HM, and BM duplexes, with only slight destabilization of base pairs, restriction of global duplex flexibility, and diminution of local nucleobase mobility. Moreover, significant restriction of the local motion of residues flanking the methylcytosine in the HM duplex suggested that these residues are more rigidly bound to SRA, in line with a slightly higher affinity of the HM duplex as compared to that of the NM or BM duplex. Our results are consistent with a “reader” role, in which the SRA domain scans DNA sequences for hemimethylated CpG sites without perturbation of the structure of contacted nucleotides

Mammalian Frataxin Controls Sulfur Production and Iron Entry during de Novo Fe₄S₄ Cluster Assembly

Iron–sulfur (Fe–S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe–S clusters are synthesized by the mitochondrial iron–sulfur cluster (ISC) machinery and the cytosolic iron–sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe–S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich’s ataxia. Here, by comparing the biochemical and spectroscopic properties of quaternary (ISCU/NFS1/ISD11/FXN) and ternary (ISCU/NFS1/ISD11) complexes, we show that FXN stabilizes the quaternary complex and controls iron entry to the complex through activation of cysteine desulfurization. Furthermore, we show for the first time that in the presence of iron and l-cysteine, an [Fe₄S₄] cluster is formed within the quaternary complex that can be transferred to mammalian aconitase (mACO2) to generate an active enzyme. In the absence of FXN, although the ternary complex can assemble an Fe–S cluster, the cluster is inefficiently transferred to ACO2. Taken together, these data help to unravel further the Fe–S cluster assembly process and the molecular basis of Friedreich’s ataxia

Cell cycle phase dependence of the levels of detyrosinated tubulin (deY-tub) and H4K20me3 in clones overexpressing hTTLL12 compared to control clones.

<p><i>A.</i> FACS scans of cells released from a double thymidine block at the indicated times. The times shown are those at which the cells were predominantly in S, G2/M and G1, except for the 18 hour time point. One representative synchronisation is shown. <i>B.</i> WBs, from one experiment, of whole cell lysates from cells synchronised in the G1, S and G2/M phases, respectively. The graphs are the averages from 3 independent experiments. * p<0.05. <i>C.</i> Proportion of cells in G1 at different times after release from double thymidine block. The average from 3 different experiments (up to 18 h) is shown. c2, c4 = DNA complements as measured by propidium iodide.</p

hTTLL12 up and down regulation prolong mitotic duration of HEp-2 cells.

<p><i>A.</i> Example frames from live cell movies of a HEp-2 cell stained with vital Hoechst. The time between nuclear envelope breakdown (NEB, first arrowhead, 0 min) and anaphase onset (second arrowhead) are indicated. Upper panel: fluorescent images (Hoechst), middle panel: phase contrast images, lower panel: overlay. <i>B–C.</i> Cumulative frequency (plot, right axis) and frequency distribution (histogram, left axis) of mitotic duration for hTTLL12 clones <i>(B)</i> and siRNA transfected HEp-2 <i>(C)</i>. <i>B.</i> hTTLL12 (hTTLL12_A-E) or Control (Control_A-E) clones were seeded in 6-well plates. 48 hours post seeding, cells were treated with vital Hoechst and analysed (Experimental Procedures). Parental HEp-2 cells (light green), and the averages of the hTTLL12 (red) and Control (dark green) clones are plotted. <i>C.</i> HEp-2 cells were transfected separately with 12.5 nM sihTTLL12_1-6 or controls (siLuciferase, siCtrl, siScramble). 72 hours post transfection, cells were stained with vital Hoechst and analysed. HEp-2 cells; light green; averages of sihTTLL12s; red; averages of siControls; dark green. More detailed information and statistical analysis can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051258#pone.0051258.s002" target="_blank">Figure S1</a>.</p

hTTLL12 alters detyrosinated tubulin levels and co-immunoprecipitates with α-tubulin.

<p><i>A.</i> hTTLL12 (hTTLL12_A-E) or Control (Control_A, _B, _D, _E) clones were harvested in Laemmli buffer and analysed by SDS-PAGE and WB. Representative WBs (left part) for detyrosinated tubulin (deY-tub) and TBP (40 µg total protein/lane; Control_D and hTTLL12_B shown). deY-tub levels from similar WBs were quantified by densitometry and normalised to TBP (right part). Data represent average detyrosinated tubulin levels ± SEM in hTTLL12 lysates relative to average levels in control lysates (n = 2). * Statistically significant difference to the controls (<i>P</i><0.05, Student's <i>t</i>-test). <i>B.</i> HEp-2 cells were transfected with 10 nM control siRNAs (siLuciferase, siCtrl, siGFP or siSilencer), 10 nM hTTLL12-specific siRNA (sihTTLL12_1-6), or 10 nM positive control siRNA (siTTL_1-2). 72 hours after transfection, cells were lysed in Laemmli buffer and analysed by SDS-PAGE and WB. Chemiluminescent signals were detected using a Versadoc image station. Representative WBs (left part) for detyrosinated tubulin (40 µg of total protein/lane) and TBP signals (note that lanes 1–3 of the TBP blot are identical to the ones shown in the upper left part of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051258#pone-0051258-g002" target="_blank">Figure 2B</a>) in siLuciferase, siSilencer, sihTTLL12_2 and siTTL_2 lysates. Detyrosinated tubulin levels were quantified using Quantity One software and normalized to TBP (right part). Data represent average detyrosinated tubulin levels ± SEM in sihTTLL12 and siTTL lysates relative to average levels in control siRNA lysates (siControl; n = 5). * Statistically significant difference to the levels in siControl (<i>P</i><0.05, Student's <i>t</i>-test). <i>C.</i> Representative WB showing the co-immunoprecipitation of α-tubulin with Flag-hTTLL12 immunoprecipitated from the stable clone hTTLL12_A (lane 3). Similar results were obtained with 4 other clones (hTTLL12_B-E, data not shown). Parental HEp-2 cells (lane1) and a stable HEp2-clone overexpressing Flag-NudCD2 (lane 2) were used as negative controls. IgG HC: IgG heavy chain.</p

hTTLL12 lacks HMTase activity.

<p>The assays included histones purified from calf thymus (<i>A</i>, lanes 2, 3,5, 6, 8–11; <i>B</i> lanes 1, 3, 5–9, 11) as substrate and proteins to be tested for HMT activity that were purified from bacterial, viral and mammalian cell expression systems. The upper panels in each part (<i>A</i> & <i>B</i>) are Coomassie Blue stained SDS-polyacrylamide gels of the expressed proteins and the lower panels are the corresponding fluorograms centred on the core histones (Histones). The proteins tested for HMTase activity were expressed and purified from appropriate pGEX vector transformed E. coli [GST-NSD1 (1700–1987) (G-NSD1), GST-TTLL12 (50–250) (G-TTLL12)], recombinant vaccinia virus Ankara strain (MVA) infected mammalian cells [vTTLL12, vTTLL12 and vTTLL12 + E (a fraction that contains EEF1A1)] and HEp-2 cell clones transformed with pSG5-puro-Flag (CON-D) or pSG5-puro-Flag TTLL12 (L12-C & L-12-D). The HEp-2 proteins were purified from cytoplasmic (c-CON-D, c-L12-c, c-L12-D), nuclear (n-CON-D, n-L12-C, n-L12-D) and 1M KCl (1M-L12-C) fractions. 10 µl reactions were loaded on 15% SDS-PAGE gels. In <i>A</i>, the approximate amounts of protein used per reaction were: GST-NSD1 (1700–1987) (1 µg, lanes 1, 2; 0.5 µg, lane 3), GST-TTLL12 (50–250) (0.5 µg, lanes 4, 5; 0.25 µg, lane 6), TTLL12 (3 µg, lanes 7, 8; 1.5 µg lane 9), TTLL12 + EEF1A1 (2 µg, lanes 10, 12) and TTL (4 µg, lane 11). In <i>B</i>, they were GST-NSD1 (1700–1987) (2 µg, lanes 1, 12), TTLL12 (0.25 µg, lanes 4–6; 0.005 µg lanes 8–10, 0.001 µg lane 11). TTLL12 was not detected in the equivalent fractions purified from the control HEp-2 clone (CON-D). In <i>B</i>, lanes 2–11, the band migrating slightly faster than GST-NSD1 (1700–1987)) is IgH from the affinity column that is eluted under the harsh denaturing conditions used for sample preparation for SDS-PAGE.</p

hTTLL12, domain organisation and similarity with other proteins.

<p><i>A.</i> Schematic representation. Red indicates the SET-like domain (amino acids 91–249), blue the TTL-like domain (amino acids 353–642), yellow or grey the predicted ATP and Mg²⁺ binding amino acid motifs that are respectively conserved (WICK^416–419, SKYI^450–453, DIRY^470–473, EVN^605–607), or non-conserved (SLDT^426–429 and RAMYAVD^578–584) in hTTLL12. <i>B, C.</i> Phylogenetic trees of the TTL domains of the human TTL family based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051258#pone.0051258.s011" target="_blank">Alignment S1 </a><i>(B)</i> and the SET-like domain of hTTLL12 and human SET domains based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051258#pone.0051258.s012" target="_blank">Alignment S2 </a><i>(C)</i>. Bootstrap values are provided for significant nodes when they are >80%. Multiple sequence alignments are available as data.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p