Mechanisms governing the accessibility of DNA damage proteins to constitutive heterochromatin.

Chromatin is thought to regulate the accessibility of the underlying DNA sequence to machinery that transcribes and repairs the DNA. Heterochromatin is chromatin that maintains a sufficiently high density of DNA packing to be visible by light microscopy throughout the cell cycle and is thought to be most restrictive to transcription. Several studies have suggested that larger proteins and protein complexes are attenuated in their access to heterochromatin. In addition, heterochromatin domains may be associated with phase separated liquid condensates adding further complexity to the regulation of protein concentration within chromocenters. This provides a solvent environment distinct from the nucleoplasm, and proteins that are not size restricted in accessing this liquid environment may partition between the nucleoplasm and heterochromatin based on relative solubility. In this study, we assessed the accessibility of constitutive heterochromatin in mouse cells, which is organized into large and easily identifiable chromocenters, to fluorescently tagged DNA damage response proteins. We find that proteins larger than the expected 10 nm size limit can access the interior of heterochromatin. We find that the sensor proteins Ku70 and PARP1 enrich in mouse chromocenters. At the same time, MRE11 shows variability within an asynchronous population that ranges from depleted to enriched but is primarily homogeneously distribution between chromocenters and the nucleoplasm. While larger downstream proteins such as ATM, BRCA1, and 53BP1 are commonly depleted in chromocenters, they show a wide range of concentrations, with none being depleted beyond approximately 75%. Contradicting exclusively size-dependent accessibility, many smaller proteins, including EGFP, are also depleted in chromocenters. Our results are consistent with minimal size-dependent selectivity but a distinct solvent environment explaining reduced concentrations of diffusing nucleoplasmic proteins within the volume of the chromocenter.

RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair.

Ring1-YY1-binding protein (RYBP) is a member of the non-canonical polycomb repressive complex 1 (PRC1), and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF) domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs), we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR) repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP) inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding.

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice.

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI).

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 µm, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin.

Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.

A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks.

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity.

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0) after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1). Mdc1 accumulates faster (T(0) = 17 ± 2 s, τ(1) = 98 ± 11 s) than 53BP1 (T(0) = 77 ± 7 s, τ(1) = 310 ± 60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T(0) = 73 ± 16 s, τ(1) = 1050 ± 270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1) of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.

GAS41 amplification results in overexpression of a new spindle pole protein.

Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.

Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4.

Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.

Quantitative analysis of DNA-damage response factors after sequential ion microirradiation.

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.