Nuclear architecture explored by live-cell fluorescence microscopy using laser and ion microbeam irradiation.

Nuclear architecture is a biological field of research that studies the spatio-temporal organization of the components within cell nuclei. Since nuclei are the organelles that harbor the genome and epigenome, they are the place where most of the genetic processes like replication, transcription, splicing, gene-regulation, DNA repair, re- combination etc. are carried out. In the presented doctoral thesis modern 4D live-cell microscopy in combination with laser or ion microbeam irradiation (to label or damage chromatin, respectively) was used to study nuclear architecture in living cells over extended periods of time at the single cell level. The results presented in this thesis can be partitioned into three main parts: (a) chromatin dynamics in cycling cells, (b) adaptation of the ion micro beam facil- ity SNAKE to the needs of live-cell observation (including first experiments) and (c) exploring spatio-temporal dynamics of DNA repair proteins after laser micro ir- radiation. (A) Chromatin dynamics in cycling cells Distribution of interphase chromosomes within cell nuclei has been found to be non- random with respect to gene density and chromosome size. Changes in nuclear orga- nization have been reported in several disorders and diseases. To which extent relative chromosome positioning is conserved through mitosis in cycling cells and whether certain chromatin domains are able change their relative position dramatically in the interphase nucleus has been the subject of various mechanistic models and contro- versial discussions. In 1909 German biologist theodor Boveri was the first one to comment on this topic in his publication: “Die Blastomerenkerne von Ascaris mega- locephala und die Theorie der Chromosomenindividualität” (included as an appendix to this thesis). In order to test Boveri’s hypotheses, 4D live-cell observations were carried out on a modern spinning disc confocal microscope using a human cell line that possesses photoactivatable chromatin. In experiments that used photoactivation and photobleaching of chromatin, it could be demonstrated that – as stated by Boveri – chromatin proximity relationships are in general not conserved through mitosis but destroyed during early prometaphase by the mechanics of mitosis. Other experiments showed that nuclear rotations in a conveyer-belt-like manner are able to bring initially distant chromatin domains into close proximity in a matter of a few minutes. (B) Adaptation of the SNAKE micro beam facility to the needs of live- cell microscopy (including first experiments) Since ordinary irradiation sources lack the ability to perform targeted micro irradia- tion at the micrometer scale and laser micro irradiation produces an artificial mix of various DNA damages, the ion microbeam SNAKE represents an interesting tool to explore the dynamics of repair proteins in a spatio-temporal context. In the course of a collaboration project the ion microbeam was adapted to the needs of long-term live-cell microscopy. These adaptations and first live-cell experiments performed at the refurbished ion micro beam are described in this part of the results. (C) Exploring spatio-temporal dynamics of DNA repair proteins after laser micro irradiation. Mutation of genetic information can cause serious harm to a cell or even a whole or- ganism. DNA repair serves to protect and clean the genome from undirected poten- tially hazardous changes. Compared to the wealth of information which is available about DNA repair at the molecular level only little attention has been payed to it in context of nuclear architecture. In the last part of the results cells stably expressing GFP tagged versions of the repair proteins MDC1, Rad52 and 53BP1 were damaged by laser micro irradiation and imaged over extended periods of time. It could be de- monstrated that at the used damage induction conditions most of the cells show only minor changes with respect to localization of damage signals, kinetochores and nu- cleoli pattern over time. Furthermore, disappearance of spontaneous 53BP1-GFP foci in favor of protein recruitment to damaged chromatin and mutual exclusion between kinetochore signals and Rad52-GFP damage foci could be observed. In a few U2OS Rad52-GFP nuclei DNA damage foci disappeared simultaneously after a dramatic phase in which the total number of foci drastically increased – even adjacent to the laser damaged chromatin

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

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less‐compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co‐aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments

4D chromatin dynamics in cycling cells: Theodor Boveri's hypotheses revisited

This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements

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

Summary: 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. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modificatio

The 4D nucleome: Evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments

AbstractRecent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC). The INC comprises the compact, transcriptionally inactive core of chromatin domain clusters (CDCs). The ANC is formed by the transcriptionally active periphery of CDCs, called the perichromatin region (PR), and the interchromatin compartment (IC). The IC is connected to nuclear pores and serves nuclear import and export functions. The ANC is the major site of RNA synthesis. It is highly enriched in epigenetic marks for transcriptionally competent chromatin and RNA Polymerase II. Marks for silent chromatin are enriched in the INC. Multi-scale cross-correlation spectroscopy suggests that nuclear architecture resembles a random obstacle network for diffusing proteins. An increased dwell time of proteins and protein complexes within the ANC may help to limit genome scanning by factors or factor complexes to DNA exposed within the ANC

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

Summary: Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2–10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from 300 Mbp/μm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC