γH2AX Foci Form Preferentially in Euchromatin after Ionising-Radiation

BACKGROUND: The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed gammaH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of gammaH2AX foci following X-irradiation with regions of the nucleus devoid of foci. METHODOLOGY/PRINCIPLE FINDINGS: Using immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication. CONCLUSIONS/SIGNIFICANCE: We discuss a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since gammaH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential responses of heterochromatic and euchromatic compartments of the genome to DSBs will have implications for understanding the processes of DNA repair in relation to nuclear and chromatin organization

An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions.

The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1α, β, and γ, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1α and, to a lesser extent, HP1γ have a closer functional link to Suv39h1, compared to HP1β. HP1α and γ preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1α stability compared HP1β and γ. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1α and γ isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1α and γ,  and enhances HP1β levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization

Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3-H4 histones and lamin-B receptor

Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1β intrabody sequesters HP1β into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1β:LBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organisation

Observation of Bs0→J/ψf0(980)B_s^0\to J/\psi f_0(980) and Evidence for Bs0→J/ψf0(1370)B_s^0\to J/\psi f_0(1370)

We report the first observation of $B_s^0\to J/\psi f_0(980)$ and first evidence for $B_s^0\to J/\psi f_0(1370)$, which are CP eigenstate decay modes. These results are obtained from $121.4\;\mathrm{fb}^{-1}$ of data collected at the $\Upsilon(5S)$ resonance with the Belle detector at the KEKB $e^+e^-$ collider. We measure the branching fractions $\mathcal{B}(B_s^0\to J/\psi f_0(980);f_0(980)\to\pi^+\pi^-)=(1.16^{+0.31}_{-0.19}(\mathrm{stat.})^{+0.15}_{-0.17}(\mathrm{syst.})^{+0.26}_{-0.18}(N_{B_s^{(*)}\bar B_s^{(*)}})) \times 10^{-4}$ with a significance of $8.4\sigma$, and $\mathcal{B}(B_s^0\to J/\psi f_0(1370);f_0(1370)\to\pi^+\pi^-)=(0.34^{+0.11}_{-0.14}(\mathrm{stat.})^{+0.03}_{-0.02}(\mathrm{syst.})^{+0.08}_{-0.05}(N_{B_s^{(*)}\bar B_s^{(*)}})) \times 10^{-4}$ with a significance of $4.2\sigma$. The last error listed is due to uncertainty in the number of produced $B_s^{(*)}\bar B_s^{(*)}$ pairs.Comment: 5 pages, 2 figures, 2 tables, published in PR

Measurements of Branching Fractions and Time-dependent CP Violating Asymmetries in B0→D(∗)±D∓B^{0} \to D^{(*)\pm}D^{\mp} Decays

We report measurements of branching fractions and time-dependent CP asymmetries in $B^{0} \to D^{+}D^{-}$ and $B^{0} \to D^{*\pm}D^{\mp}$ decays using a data sample that contains $(772 \pm 11)\times 10^6 B\bar{B}$ pairs collected at the $\Upsilon(4S)$ resonance with the Belle detector at the KEKB asymmetric-energy $e^+ e^-$ collider. We determine the branching fractions to be $\mathcal{B}(B^{0} \to D^{+}D^{-})=(2.12 \pm 0.16 \pm 0.18)\times 10^{-4}$ and $\mathcal{B}(B^{0} \to D^{*\pm}D^{\mp}=(6.14 \pm 0.29 \pm 0.50)\times 10^{-4}$. We measure CP asymmetry parameters $\mathcal{S}_{D^{+}D^{-}} = -1.06_{-0.14}^{+0.21} \pm 0.08$ and $\mathcal{C}_{D^{+}D^{-}} = -0.43 \pm 0.16 \pm 0.05$ in $B^{0} \to D^{+}D^{-}$ and $\mathcal{A}_{D^{*}D} = +0.06 \pm 0.05 \pm 0.02$, $\mathcal{S}_{D^{*}D} = -0.78 \pm 0.15 \pm 0.05$, $\mathcal{C}_{D^{*}D} = -0.01 \pm 0.11 \pm 0.04$, $\Delta\mathcal{S}_{D^{*}D} = -0.13 \pm 0.15 \pm 0.04$ and $\Delta\mathcal{C}_{D^{*}D} = +0.12 \pm 0.11 \pm 0.03$ in $B^{0} \to D^{*\pm}D^{\mp}$, where the first uncertainty is statistical and the second is systematic. We exclude the conservation of CP symmetry in both decays at equal to or greater than $4\sigma$ significance.Comment: 7 pages, 2 figure

Evidence for B- -> tau- nu_bar with a Semileptonic Tagging Method

We present a measurement of the decay B- -> tau- nu_bar using a data sample containing 657 million BB_bar pairs collected at the Upsilon(4S) resonance with the Belle detector at the KEKB asymmetric-energy e+e- collider. A sample of BB_bar pairs are tagged by reconstructing one B meson decaying semileptonically. We detect the B- -> tau- nu_bar candidate in the recoil. We obtain a signal with a significance of 3.6 standard deviations including systematic uncertainties, and measure the branching fraction to be Br(B- -> tau- nu_bar) = [1.54+0.38-0.37(stat)+0.29-0.31(syst)]*10^-4. This result confirms the evidence for B- -> tau- nu_bar obtained in a previous Belle measurement that used a hadronic B tagging method.Comment: 7 pages, 3 figures, corrected references, to appear in PRD-R