Kumiko Samejima

Trashing the genome: the role of nucleases during apoptosis

Two classes of nucleases degrade the cellular DNA during apoptosis. Cell-autonomous nucleases cleave DNA within the dying cell. They are not essential for apoptotic cell death or the life of the organism, but they might affect the efficiency of the process. By contrast, waste-management nucleases are essential for the life of the organism. In post-engulfment DNA degradation, the DNA of apoptotic cells is destroyed in lysosomes of the cells that have phagocytosed the corpses. Waste-management nucleases also destroy DNA that is released into the extracellular compartment. Here, we describe the complex group of nucleases that are involved in DNA destruction during apoptotic cell death.

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

During the shaping of mitotic chromosomes, KIF4 and condensin work in parallel to promote lateral chromatid compaction and in opposition to topoisomerase IIα, which shortens the chromatid arms.

DNA topoisomerase IIα interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.

INCENP–aurora B interactions modulate kinase activity and chromosome passenger complex localization

Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Apoptosis-associated caspase activation assays

Caspases are aspartate-directed cysteine proteases that cleave a diverse group of intracellular substrates to contribute to various manifestations of apoptosis. These proteases are synthesized as inactive precursors and are activated as a consequence of signaling induced by a wide range of physiological and pathological stimuli. Caspase activation can be detected by measurement of catalytic activity, immunoblotting for cleavage of their substrates, immunolabeling using conformation-sensitive antibodies or affinity labeling followed by flow cytometry or ligand blotting. Here we describe methods for each of these assays, identify recent improvements in these assays and outline the strengths and limitations of each approach.

Detection of DNA cleavage in apoptotic cells.

At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.

Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery

When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells. DOI: http://dx.doi.org/10.7554/eLife.01641.001

A pathway for mitotic chromosome formation

Tracking mitotic chromosome formation How cells pack DNA into fully compact, rod-shaped chromosomes during mitosis has fascinated cell biologists for more than a century. Gibcus et al. delineated the conformational transition trajectory from interphase chromatin to mitotic chromosomes minute by minute during the cell cycle. The mitotic chromosome is organized in a spiral staircase architecture in which chromatin loops emanate radially from a centrally located helical scaffold. The molecular machines condensin I and II play distinct roles in these processes: Condensin II is essential for helical winding, whereas condensin I modulates the organization within each helical turn. Science, this issue p. eaao6135 Mitotic chromosome folding involves formation of increasingly compacted helically arranged nested loop arrays. INTRODUCTION During mitosis, cells compact their chromosomes into dense rod-shaped structures to ensure their reliable transmission to daughter cells. Our work explores how cells achieve this compaction. We integrate genetic, genomic, and computational approaches to characterize the key steps in mitotic chromosome formation from the G2 nucleus to metaphase, and we identify roles of specific molecular machines, condensin I and II, in these major conformational transitions. RATIONALE We used chicken DT-40 cells expressing an analog-sensitive CDK1 to produce cell cultures that synchronously enter mitosis. We collected cells at key time points during mitotic entry; analyzed chromosome organization by microscopy, chromosome conformation capture, and polymer simulations; and delineated a pathway of mitotic chromosome formation. We used engineered cell lines to study the function of condensin complexes, which are critical for mitotic chromosome formation. We fused condensin I and II subunits to plant auxin-inducible degron domains, thus enabling their rapid depletion in late G2 just before mitotic entry. These cell lines allowed us to determine the roles of condensin I and II in specific steps of the mitotic chromosome morphogenesis pathway. RESULTS Our analysis of G2 chromosomes reveals hallmarks of interphase chromosomes, including topologically associating domains and compartments. Upon entry into prophase, this organization is lost within minutes, and by late prophase, chromosomes are folded as arrays of consecutive loops condensed around a central axis. These loops project with random but mutually correlated angles from the axis. During prometaphase, the loop array undergoes two major reorganizations. First, it acquires a helical arrangement of loops. Polymer simulations of Hi-C data show that the centrally located axis acquires a helical twist so that consecutive loops emanate as the steps of a spiral staircase. Second, the chromatin loops become nested with ~400-kb outer loops split up by ~80-kb inner loops. As prometaphase proceeds, chromosomes shorten through progressive helical winding, with the numbers of loops per turn increasing. As a result, the size of a helical turn grows from ~3 Mb (~40 loops) to ~12 Mb (~150 loops). Depletion of condensin I or II before mitotic entry revealed their differing roles in mitotic chromosome formation. Either condensin can mediate loop array formation. However, condensin II is required for the helical twisting of the scaffold from which loops emanate, whereas condensin I modulates the size and arrangement of nested inner loops. CONCLUSION We describe a pathway of mitotic chromosome folding that unifies many previous observations. In prophase, condensins mediate the loss of interphase organization and the formation of arrays of consecutive loops. In prometaphase, chromosomes adopt a spiral staircase–like structure with a helically arranged axial scaffold of condensin II at the bases of chromatin loops. The condensin II loops are further compacted by condensin I into clusters of smaller nested loops that are additionally collapsed by chromatin-to-chromatin attractions. The combination of nested loops distributed around a helically twisted axis plus dense chromatin packing achieves the 10,000-fold compaction of chromatin into linearly organized chromosomes that is required for accurate chromosome segregation when cells divide. A pathway for mitotic chromosome formation. In prophase, condensins mediate the loss of interphase chromosome conformation, and loop arrays are formed. In prometaphase, the combined action of condensin I (blue spheres in the bottom diagram) and II (red spheres) results in helically arranged nested loop arrays. Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central “spiral staircase” condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.

The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis

Background: INCENP is predicted to have a coiled coil domain. Results: The coil is actually a stable single α-helix (SAH) domain that is highly extendable and directly binds microtubules. Conclusion: This flexible dog leash may allow Aurora B to associate with dynamic targets in the outer kinetochore. Significance: The SAH domain allows CPC flexibility without requiring complex dimerization. The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible “dog leash,” allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes

We describe a method for the isolation of conditional knockouts of essential multiply spliced genes in which the entire body of the gene downstream of the ATG start codon is left untouched but can be switched off rapidly and completely by adding tetracycline to the culture medium. The approach centers on a “promoter-hijack” strategy in which the genes promoter is replaced with a minimal promoter responsive to the tetracycline-repressible transactivator (tTA). Elsewhere in the genome, a cloned fragment of the genes promoter is used to drive expression of a tTA. Thus, the gene is essentially regulated by its own promoter but through the intermediary tTA. Using this strategy, we generated a conditional knockout of chromokinesin KIF4A, an important mitotic effector protein whose mRNA is multiply spliced and whose cDNA is highly toxic when overexpressed in cells. We used chicken DT40 cells, but the same strategy should be applicable to ES cells and, eventually, to mice.