Reduced Mobility of the Alternate Splicing Factor (Asf) through the Nucleoplasm and Steady State Speckle Compartments

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF–GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF–GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites

Heterochromatin is refractory to γ-H2AX modification in yeast and mammals

Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (γ-H2AX). In budding yeast, a single endonuclease-induced DSB triggers γ-H2AX modification of 50 kb on either side of the DSB. The extent of γ-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of γ-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of γ-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a γ-H2AX–covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, γ-H2AX distribution shows that γ-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive γ-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs

Chimeric IgH-TCR/ translocations in T lymphocytes mediated by RAG

Translocations involving the T cell receptor alpha/delta (TCRα/δ) chain locus, which bring oncogenes in the proximity of the TCRα enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRα/δ locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRα/δ associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRα/δ fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRα/δ as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs

Alterations in the Chromatin Environment Following the Introduction of DNA Breaks

The presence of DNA breaks has extensive biochemical implications for the integrity of the genome. It is well established that distinct DNA damage response proteins are recruited to, and accumulate at, sites of genomic lesions, including kinases that initiate multiple DNA damage signaling cascades. The repair of DNA breaks is facilitated by the phosphorylation of H2AX, which organizes DNA damage response factors in the vicinity of the lesion. Metabolism of the DNA breaks occurs in a chromatin environment and modulating chromatin structure is necessary for the fidelity of the DNA damage response. We set out to determine in living cells both how chromatin is remodeled in the presence of DNA breaks and whether the establishment of large sub-cellular DNA damage response domains influences other DNA metabolic processes, such as transcription. Using a photoactivatable histone H2B, we examined the mobility and structure of chromatin immediately after the introduction of DNA breaks. We find that chromatin-containing damaged DNA exhibits limited mobility but undergoes an initial energy-dependent local expansion that occurs independently of H2AX and ATM. We also took advantage of the large copy number, tandem gene arrangement, and spatial organization of ribosomal transcription units as a model system to measure the kinetics of transcription in real time in the presence of DNA breaks. We find that RNA polI inhibition is not the direct result of the physical DNA break but mediated by ATM kinase activity and surrogate DNA repair proteins. We propose that the localized opening of chromatin at DNA breaks establishes an accessible biochemically unique sub-nuclear environment that facilitates DNA damage signaling and repair

Synthesis, Characterization, and Direct Intracellular Imaging of Ultrasmall and Uniform Glutathione‐Coated Gold Nanoparticles

Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and how they behave in the body. Here we report the synthesis of ultrasmall, uniform 144‐atom AuNPs protected by p ‐mercaptobenzoic acid followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH‐coated nanoparticles (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles’ intracellular fate, the cell‐penetrating peptide TAT is attached noncovalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)‐TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells. The synthesis and characterization of an ultrasmall and uniform glutathione‐coated gold nanoparticle is reported. It is also shown that scanning transmission electron microscopy (STEM) enables the visualization and quantification of individual gold nanoparticles as well as small aggregates in the cytoplasm and nucleus of HeLa cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92372/1/2277_ftp.pd

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.We thank Anthony Tubbs for comments on the paper; Jennifer Mehalko and Dom Esposito (Protein Expression Laboratory, Frederick National Laboratory for Cancer Research) for transgenic constructs; Karim Baktiar, Diana Haines, and Elijah Edmonson (Pathology/Histotechnology Laboratory, Frederick National Laboratory for Cancer Research) for rodent necropsy, pathology analysis, and imaging; Joseph Kalen and Nimit Patel (Small Animal Imaging Program, Frederick National Laboratory for Cancer Research) for X-ray computed tomography (CT) scan imaging; Jennifer Wise and Kelly Smith for assistance with animal work; Davide Robbiani and Kai Ge for antibodies; Dan Durocher for shieldin constructs; David Goldstein and the CCR Genomics core for sequencing support; and Neil Johnson for discussions. Research in the J.M.S. laboratory is supported by NIH grant R01CA197506. Research in the N.M. laboratory is supported by NIH grant R01 227001. The A.N. laboratory is supported by the Intramural Research Program of the NIH, an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11), the Department of Defense Idea Expansion (W81XWH-15-2-006) and Breakthrough (W81XWH-16-1-599) Awards, the Alex's Lemonade Stand Foundation Award, and an NIH Intramural FLEX Award.S

Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation