End Crisis, Bridges and Scattered Genes: Chromatin Bridges and their Role in Genomic Stability

By Gesa Junge, PhD

Each of our cells contains about two meters of DNA which needs to be stored in cells that are often less than 100uM in diameter, and to make this possible, the DNA is tightly packed into chromosomes. As the human cell prepares to divide, the 23 pairs of chromosomes neatly line up and attach to the spindle apparatus via their middle point, the centrosome. The spindle apparatus is part of the cell’s scaffolding and it pulls the chromosomes to opposite ends of the cell as the cell divides, so that every new daughter cell ends up with exactly one copy of each chromosome. This is important; cells with more or less than one copy of a chromosome are called aneuploid cells, and aneuploidy can lead to genetic disorders such as Down Syndrome (three copies of chromosome 21).

In some cancer cells, chromosomes with two centromeres (dicentric chromosomes) can be detected, which can happen when the ends of two chromosomes fuse in a process called telomere crisis. Telomeres are a sort of buffer zone at the ends of the chromosome which consist of repeats of non-coding DNA sequences, meaning there are no genes located here. As one of the DNA strands is not replicated continuously but in fragments, the telomeres get shorter over the lifespan of a cell, and short telomeres can trigger cell cycle arrest before the chromosomes get so short that genetic information is lost. But occasionally, and especially in cancer cells, chromosome ends fuse and a chromosome becomes dicentric. Then it can attach to the spindle apparatus in two points and may end up being pulled apart as the two daughter cells separate, sort of like a rope tied to two cars that drive in opposite directions. This string of chromosome is referred to as a chromatin bridge.

Researchers at Rockefeller University are studying these chromatin bridges and what their relevance is for the health of the cell. A paper by John Maciejowski and colleagues found that the chromatin bridges actually stay intact for quite a long time. Chromosomes are pretty stable, and so the chromatin bridges lasted for an average of about 9 hours (3-20h) before snapping and quickly being pulled back into the original cell (see video). Also, the nucleus of the cell was often heart-shaped as opposed to the usual round shape, which suggests that the chromatin bridge physically pulls on the membrane surrounding the nucleus, the nuclear envelope. Indeed, proteins that make up the nuclear envelope (e.g. LAP2) were seen on the chromatin bridge, suggesting that they take part of the nuclear envelope with them as they divide.  Also, cells with chromatin bridges had temporary disruptions to their nuclear envelope at some point after the bridge was resolved, more so than cells without chromatin bridges.

The chromatin bridges also stained positive for replication protein A (RPA), which binds single stranded DNA. DNA usually exists as two complementary strands bound together, and the two strands really only separate to allow for DNA to be copied or transcribed to make protein. Single-stranded DNA is very quickly bound by RPA, which stabilises it so it does not loop back on itself and get tangled up in secondary structures. The Rockefeller study showed that a nuclease, a DNA-eating enzyme, called TREX1 is responsible for generating the single-stranded DNA on chromatin bridges. And this TREX1 enzyme seems to be really important in resolving the chromatin bridges: cells that do not have TREX1 resolve their chromatin bridges later than cells that do have TREX1.

So how are chromatin bridges important for cells, the tissue and the organism (i.e. us)? The authors of this study suggest that chromatin bridges can lead to a phenomenon called chromothripsis. In chromothripsis, a region of a chromosome is shattered and then put back together in a fairly random order and with some genes facing the wrong direction. Think of a new, neatly color-sorted box of crayons that falls on the floor, and then someone hastily shoves all the crayons back in the box with no consideration for color coordination or orientation. Chromothripsis occurs in several types of cancers, but it is still not really clear how often, in what context and exactly how the genes on a chromosome end up in such a mess.

According to this study, chromothripsis may be a consequence of telomere crisis, and chromatin bridges could be part of the mechanism: A chromosome fuses ends with another chromosome and develops two centromeres. The dicentric chromosome attaches to two opposite spindles and is pulled apart during cell division, generating a chromatin bridge which is attacked by TREX that turns it into single-stranded DNA, the bridge snaps and in the process the DNA scatters, and returns to the parent cell where it is haphazardly reassembled, leaving a chromothripsis region.

The exact mechanisms of this still need to be studied and the paper mentions a few important discussion points. For example, all the experiments were performed in cell culture, and the picture may look very different in a tumor in a human being. And what exactly causes the bridge to break? Also, there are probably more than one potentially mechanism linking telomere crisis to chromothripsis. But it is a very interesting study that shines some light on the somewhat bizarre phenomenon of chromothripsis, and the importance of telomere crisis.

Reference: Maciejowski et al, Cell. 2015 Dec 17; 163(7): 1641–1654.

 

 

The Phase That Makes The Cell Go Round

 

By Johannes Buheitel, PhD

 

There comes a moment in every cell’s life, when it’s time to reproduce. For a mammalian cell, this moment usually comes at a ripe age of about 24 hours, at which it undergoes the complex process of mitosis. Mitosis is one of the two main chromosomal events of the cell cycle. But in contrast to S phase (and also to the other phases of the cell cycle) it’s the only phase that is initiated by a dramatic change in the cell’s morphology that, granted, you can’t see with your naked eye, but definitely under any half-decent microscope without requiring any sort of tricks (like fluorescent proteins): Mitotic cells become perfectly round. This transformation however, as remarkable as it may seem, is merely a herald for the main event, which is about to unfold inside the cell: An elegant choreography of chromosomes, which crescendoes into the perfect segregation of the cell’s genetic content and the birth of two new daughter cells.

 

To better understand the challenges behind this choreography, let’s start with some numbers: A human cell has 23 unique chromosomes (22 autosomes and 1 gonosome) but since we’re diploid (each chromosome has a homolog) that brings us to a total of 46 chromosomes that are present at any given time, in (nearly) every cell of our bodies. Before S phase, each chromosome consists of one continuous strand of DNA, which is called a chromatid. Then during S phase, a second “sister” chromatid is being synthesized as a prerequisite for later chromosome segregation in M phase. Therefore, a pre-mitotic cell contains 92 chromatids. That’s a lot! In fact, if you laid down all the genetic material of a human cell that fits into a 10 micrometer nucleus, end to end on a table, you would wind up having with a nucleic acid string of about 2 meters (around 6 feet)! The challenge for mitosis is to entangle this mess and ultimately divide it into the nascent daughter cells according to the following rules: 1) Each daughter gets exactly half of the chromatids. 2) Not just any chromatids! Each daughter cell requires one chromatid of each chromosome. No more, no less. And maybe the most important one, 3) Don’t. Break. Anything. Sounds easy? Far from it! Especially since the stakes are high: Because if you fail, you die (or are at least pretty messed up)…

 

Anatomy of a mitotic chromosome
Anatomy of a mitotic chromosome

To escape this dreadful fate, mitosis has evolved into this highly regulated process, which breaks down the high complexity of the task at hand into more sizable chunks that are then dealt with in a very precise spatiotemporal manner. One important feature of chromosomes is that its two copies – or sister chromatids – are being physically held together from the time of their generation in S phase until their segregation into the daughter cells in M phase. This is achieved by a ring complex called cohesin, which topologically embraces the two sisters in its lumen (we’ll look at this interesting complex in a separate blog post). This helps the cell to always know, which two copies of a chromosome belong together, thus essentially cutting the complexity of the whole system in half, and that before the cell even enters mitosis.

Actual mitosis is divided into five phases with distinct responsibilities: prophase, prometaphase, metaphase, anaphase and telophase (cytokinesis, the process of actually dividing the two daughter cells, is technically not a phase of mitosis, but still a part of M phase). In prophase, the nuclear envelope surrounding the cell’s genetic content is degraded and the chromosomes begin to condense, which means that each DNA double helix gets neatly wrapped up into a superstructure. Think of it like taking one of those old coiled telephone receiver cables (that’s your helix) and wrapping it around your arm. So ultimately, chromosome condensation makes the chromatids more easily manageable by turning them from really long seemingly entangled threads into a shorter (but thicker) package. At this point each chromatid is still connected to its sister by virtue of the cohesin complex (see above) at one specific point, which is called the centromere. It is this process of condensation of cohesed sister chromatids that is actually responsible for the transformation of chromosomes into their iconic mitotic butterfly shape that we all know and love. While our butterflies are forming, the two microtubule-organizing centers of the cell, the centrosomes, begin to split up and wander to the cell poles, beginning to nucleate microtubules. In prometaphase, chromosome condensation is complete and the centrosomes have reached their destination, still throwing out microtubules like it’s nobody’s business. During this whole time, their job is to probe the cytoplasm for chromosomes by dynamically extending and collapsing, trying to find something to hold on to amidst the darkness of the cytoplasm. This something is a protein structure, called the kinetochore, which sits on top of each sister chromatid’s centromere region. Once a microtubule has found a kinetochore, it binds to it and stabilizes. Not all microtubules will bind to kinetochores though, some of them will interact with the cell cortex or with each other to gradually form the infamous mitotic spindle, the scaffold tasked with directing the remainder of the chromosomal ballet. Chromsomes, which are attached to the spindle (via their kinetochores) will gradually move (driven by motor proteins like kinesins) towards the middle region of the mother cell and align on an axis, which lies perpendicular between the two spindle poles. This axis is called the metaphase plate and represents a visual mark for the eponymous phase. The transition from metaphase to anaphase is the pivotal moment of mitosis; the moment, when sister chromatids become separated (by proteolytic destruction of cohesin) and subsequently move along kinetochore-associated microtubules with the help of motor proteins towards cell poles. As such a critical moment, the metaphase-to-anaphase transition is tightly safeguarded by a checkpoint, the spindle assembly checkpoint (SAC), which ensures that every single chromatid is stably attached to the correct side of the spindle (we’ll go into some more details in another blog post). In the following telophase, the newly separated chromosomes begin to decondense, the nuclear envelope reforms and the cell membrane begins to restrict in anticipation of cytokinesis, when the two daughter cells become physically separated.

 

Overview over the five phases of mitosis.
Overview over the five phases of mitosis (click to enlarge).

To recap, the process of correctly separating the 92 chromatids of a human cell into two daughter cells is a highly difficult endeavor, which, however, the cell cleverly deals with by (1) keeping sister chromatids bound to each other, (2) wrap them  into smaller packets by condensation, (3) attach each of these packets to a scaffold a.k.a. mitotic spindle, (4) align the chromosomes along the division axis, so that each sister chromatid is facing opposite cell poles, and finally (5) move now separated sister chromatids along this rigid scaffold into the newly forming daughter cells. It’s a beautiful but at the same time dangerous choreography. While there are many mechanisms in place that protect the fidelity of mitosis, failure can have dire consequences, of which cell death isn’t the worst, as segregation defects can cause chromosomal instabilities, which are typical for tissues transforming into cancer. In future posts we will dive deeper into the intricacies of the chromosomal ballet, that is the centerpiece of the cell cycle, as well as the supporting acts that ensure the integrity of our life’s code.

 

What’s Chromatin Got to Do With It?

Alisa Moskaleva

 

We know that cells lead intricate lives of growth, change, and division. We also know that DNA has not only the four letters A, T, C, and G, but also an intricate grammar of modifications on DNA-associated proteins, termed chromatin, that changes over time. We can surmise that there is a connection between the life cycle of a cell, called the cell cycle, and its chromatin. But how does the cell cycle influence chromatin? Yang Xu and colleagues shed new light on this question in a paper in the latest issue of the Cell Cycle.

 

Before a cell can divide, it must first condense its chromatin into packages called mitotic chromosomes, so that its genome may be evenly divided between its two daughters. One of the chromatin modifications that promotes this condensation is the deubiquitination of histone H2A. It’s been known for six years that a protein called Ubp-M can deubiquitinate histone H2A. Now Xu and colleagues explain what causes Ubp-M to deubiquitinate histone H2A before mitosis and not at other times in the cell cycle.

 

Xu and colleagues focused on a phosphorylation on the 552nd amino acid, a serine, of Ubp-M. This serine is in a motif that a kinase called CDK1 likes to phosphorylate. CDK1 is to the cell cycle what a conductor is to the symphony orchestra: it coordinates all the events, so that they happen in the right sequence and at the appropriate time. By knocking down CDK1 and using chemical inhibitors, Xu and colleagues established that CDK1 indeed phosphorylates Ubp-M on its serine 552.

 

Phosphorylation changes interactions between proteins. To find the function of the phosphorylation of serine 552, Xu and colleagues looked at the interaction between Ubp-M and a nuclear exporter called CRM1. This is a particularly interesting interaction because Ubp-M spends most of the cell cycle in the cytoplasm, even though it must go to the nucleus to deubiquitinate histone H2A. Therefore, Ubp-M is actively exported from the nucleus, and Xu and colleagues used an inhibitor of CRM1 to show that CRM1 participates in this export. Interestingly, a mutant version of Ubp-M that cannot be phosphorylated on the 552nd amino acid does not get exported as much. This mutant version also decreases cell proliferation and reduces the number of cells that enter mitosis. However, the mutation has no effect on the ability of Ubp-M to deubiquitinate histone H2A. Since CDK1 becomes more active before mitosis, Xu and colleagues propose that it phosphorylates Ubp-M on serine 552 and increases the fraction of Ubp-M in the nucleus, thus promoting chromatin condensation and mitosis.

Serine 552 of Ubp-M is present in primates but is not conserved in the mouse or rat homolog of Ubp-M. Though this particular example of temporal control using phosphorylation and localization occurs in only a few animal species, the principle is likely more general. Moreover, Ubp-M may contain other more conserved phosphorylation sites that function in the same way. And it is intriguing to speculate what special function this phosphorylation may serve in primates. Regardless, Xu and colleagues flesh out a direct connection between the cell cycle and chromatin modification to a rare level of detail.