One ring to rule them all: The cohesin complex

By Johannes Buheitel, PhD

In my blog post about mitosis (http://www.myscizzle.com/phases-of-mitosis/), I explained some of the challenges a human cell faces when it tries to disentangle its previously replicated chromosomes (for an overview of the cell cycle, see also http://www.myscizzle.com/cell-cycle-introduction/) and segregate them in a highly ordered fashion into the newly forming daughter cells. I also mentioned a protein complex, which is integral for this chromosomal ballet, the cohesin complex. To recap, cohesin is a multimeric ring complex, which holds the two chromatids of a chromosome together from the time the second sister chromatid is generated in S phase until their separation in M phase. This decreases complexity, and thereby increases the fidelity of chromosome segregation, and thus, mitosis/cell division. And while this feat should already be enough to warrant devoting a whole blog post to cohesin, you will shortly realize that this complex also performs a myriad of other functions during the cell cycle, which really makes it “one ring to rule them all”.

Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces ("coheses") sister chromatids.
Figure 1: The cohesin complex. The core complex consists of three subunits: Scc1/Rad21, Smc1, and Smc3. They interact to form a ring structure, which embraces (“coheses”) sister chromatids.

But let’s back up a little first. Cohesin’s integral ring structure is composed of three proteins: Smc1, Smc3 (Structural maintenance of chromosomes), and Scc1/Rad21 (Sister chromatid cohesin/radiation sensitive). These three proteins attach to each other in a more or less end-to-end manner, thereby forming a circular structure (see Figure 1; ONLY for the nerds: Smc1 and -3 form from long intramolecular coiled-coils by folding back onto themselves, bringing together their N- and C-termini at the same end. This means that these two proteins actually interact with their middle parts, forming the so-called “hinge”, as opposed to really “end-to-end”). Cohesin obviously gets its name from the fact that it causes “cohesion” between sister chromatids, which has been first described 20 years ago in budding yeast. The theory that the protein complex does so by embracing DNA inside the ring’s lumen was properly formulated in 2002 by the Nasmyth group, and much evidence supporting this “ring embrace model” has been brought forth over last decades, making it widely (but not absolutely) accepted in the field. According to our current understanding, cohesin is already loaded onto DNA (along the entire length of the decondensed one-chromatid chromosome) in telophase, i.e. only minutes after chromosome segregation, by opening/closing its Smc1-Smc3 interaction site (or “entry gate”). When the second sister chromatid is synthesized in S phase, cohesin establishes sister chromatid cohesion in a co-replicative manner (only after you have the second sister chromatid, you can actually start talking about “cohesion”). Early in the following mitosis, in prophase to be exact, the bulk of cohesin is removed from chromosome arms in a non-proteolytic manner by opening up the Smc3-Scc1/Rad21 interface (or “exit gate”; this mechanism is also called “prophase pathway”). However, a small but very important fraction of cohesin molecules, which is located at the chromosomes’ centromere regions, remains protected from this removal mechanism in prophase. This not only ensures that sister chromatids remain cohesed until the metaphase-to-anaphase transition, but also provides us with the stereotypical image of an X-shaped chromosome. The last stage in the life of a cohesin ring is its removal from centromeres, a tightly regulated process, which involves proteolytic cleavage of cohesin’s Scc1/Rad21 subunit (see Figure 2).

Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase ("entry gate"). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called "prophase pathway") by opening up the interphase between Scc1/Rad21 and Smc3 ("exit gate"). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.
Figure 2: The cohesin cycle. Cohesin is topologically loaded onto DNA in telophase by opening up the Smc1-Smc3 interphase (“entry gate”). Sister chromatid cohesion is established during S phase, coinciding with the synthesis of the second sister. In prophase of early mitosis, the bulk of cohesin molecules are removed from chromosome arms (also called “prophase pathway”) by opening up the interphase between Scc1/Rad21 and Smc3 (“exit gate”). Centromeric cohesin is ultimately proteolytically removed at the metaphase-to-anaphase transition.

As you can see, during the 24 hours of a typical mammalian cell cycle, cohesin is pretty much always directly associated with the entire genome (the exceptions being chromosomes arms during most of mitosis, i.e. 20-40 minutes and entire chromatids during anaphase, i.e. ~10 minutes). This means that cohesin has at least the potential to influence a whole bunch of other chromosomal events, like DNA replication, gene expression and DNA topology. And you know what? Turns out it does!

Soon after cohesin was described as this guardian of sister chromatid cohesion, it also became clear that there is just more to it. Take DNA replication for example. There is good evidence that initial cohesin loading is already topological (meaning, the ring closes around the single chromatid). That poses an obvious problem during S phase: While DNA replication machineries (“replisomes”) zip along the chromosomes trying to faithfully duplicate the entire genome in a matter of just a couple of hours, they encounter – on average – multiple cohesin rings that are already wrapped around DNA. Simultaneously, cohesin’s job is to take those newly generated sister chromatids and hold them tightly to the old one. Currently, we don’t really know how this works, whether the replisome can pass through closed cohesin rings, or whether cohesin gets knocked off and reloaded after synthesis. What we do know, however, is that cohesion establishment and DNA replication are strongly interdependent, with defects in cohesion metabolism causing replication phenotypes and vice versa.

Cohesin has also been shown to have functions in transcriptional regulation. It was observed quite early that cohesin can act as an insulation factor, blocking long-range promoter-enhancer association. Today we have good evidence showing that cohesin binds to chromosomal insulator elements that are usually associated with the CTCF (CCCTC-binding factor) transcriptional regulator. Here, the ring complex is thought to help CTCF’s agenda by creating internal loops, i.e. inside the same sister chromatid!

Studying cohesin has, of course, not only academic value. Because of its pleiotropic functions, defects in human cohesin biology can cause a number of clinically relevant issues. Since actual cohesion defects will cause mitotic failure (which most surely results in cell death), most of cohesin-associated diseases are believed to be caused by misregulation of the complex’s non-canonical functions in replication/transcription. These so-called cohesinopathies (e.g. Roberts syndrome and Cornelia de Lange syndrome) are congenital birth defects with widely ranging symptoms, which usually include craniofacial/upper limb deformities as well as mental retardation.

It is important to mention that cohesin also has a very unique role in meiosis where it not only coheses sister chromatids but also chromosomal homologs (the two maternal/paternal versions of a chromosome, each consisting of two sisters, which themselves are cohesed). As a reminder, the lifetime supply of all oocytes of a human female is produced before puberty. These oocytes are arrested in prophase I (prophase of the first meiotic division) with fully cohesed homologs and sisters, and resume meiosis one by one each menstrual cycle. This means that some oocytes might need to keep up their cohesion (between sisters AND homologs) over decades, which, considering the half-life of your average protein, can be challenging. This has important medical relevance as cohesion failure is believed to be the main cause behind missegregation of homologs, and thus, age-related aneuploidies, like e.g. trisomy 21.

After twenty years of research, the cohesin complex still manages to surprise us regularly, as new functions in new areas of cell cycle regulation come to light. Currently, extensive research is conducted to better understand the role of certain cohesin mutations in cancers such as glioblastoma, or Ewing’s sarcoma. And while we’re still far away from completely understanding this complex complex, we already know enough to say that cohesin really is “one ring to rule them all”.

 

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.

 

Chromosome Silencing Offers New Insights into Down Syndrome

Sally Burn

Down syndrome is caused by the most common chromosomal abnormality in live-born humans: Trisomy 21. Individuals with Down syndrome have three copies (trisomy) of chromosome 21 instead of the usual two. This excess of genetic information leads to deviations in embryonic development such that the baby is born with a subset of defects from a spectrum of characteristic traits. The most obvious indicators are the distinctive Down syndrome facial features (almond shaped eyes, small ears, large tongue) and abnormalities of the hands (single crease on palm, small curved pinky fingers). Further examination may then reveal more serious medical problems including heart abnormalities, gastrointestinal defects, and impaired vision and hearing. Intellectual disabilities are also a common problem. Continue reading “Chromosome Silencing Offers New Insights into Down Syndrome”

Mysteries of Aneuploidy

Nicole Crown

I’ve often wondered why humans are so bad at reproduction.  It’s been estimated that 10-30% of all fertilized eggs are aneuploid, and approximately one third of all miscarriages are due to aneuploidy (Hassold et al.).  In striking contrast, only 1-2% of fertilized eggs are aneuploid in mice.  As a researcher that studies meiosis, this baffles me.  The meiotic program is astoundingly complex, coordinating the repair of programmed DNA damage, finding and pairing homologous chromosomes, along with attaching the spindle in the correct orientation.  This complexity requires multiple checkpoints throughout the process to ensure everything is going as planned; if something goes wrong, the checkpoint stops the cell. Given multiple opportunities to ensure chromosomes are properly segregated, how is it that, in humans, aneuploid cells not only make it through a complete meiosis, but also go on to complete oogenesis?

Recent work from Dokshin et al. suggests that one potential way this can happen is to decouple oogenesis from meiosis.  In mice, early germ line cells initiate meiosis after Stra8 expression is turned on by a retinoic acid signal.  In the absence of Stra8, cells never initiate meiosis, and by 6-8 weeks of age, the ovaries contain no germ cells. However, a very small percentage of cells are able to escape the Stra8 phenotype, and while they still don’t initiate meiosis, they do proceed through oogenesis.  These “oocyte-like cells”, as the authors call them, have the same morphological and physiological characteristics as normal oocytes: they are able to make a zona pellucida, generate follicles, can be ovulated and fertilized, and the embryo can undergo the first division.  When the authors looked at the chromosome complement of the oocyte-like cells, they found that the chromosomes were randomly distributed between the polar body and the oocyte-like cell.

Certainly, these data show that in the absence of meiosis, the vast majority of female germ cells will not differentiate; therefore, there must be some way of monitoring the meiotic state of a cell before oocyte differentiation begins.  However, this monitoring clearly can fail, as the Stra8 deficient mice do produce oocyte-like cells capable of being fertilized.  The authors suggest some cases of human infertility may be explained by a disconnect between oocyte differentiation and meiosis.  It will be fascinating to compare the mechanism of communication between meiotic state and oocyte differentiation in humans to other organisms, and determine if the apparently higher rate of aneuploidy in humans is sometimes due to miscommunication.