One ring to rule them all: The cohesin complex

By Johannes Buheitel, PhD

In my blog post about 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 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”.


HeLa, the VIP of cell lines

By  Gesa Junge, PhD

A month ago, The Immortal Life of Henrietta Lacks was released on HBO, an adaptation of Rebecca Skloot’s 2010 book of the same title. The book, and the movie, tell the story of Henrietta Lacks, the woman behind the first cell line ever generated, the famous HeLa cell line. From a biologist’s standpoint, this is a really unique thing, as we don’t usually know who is behind the cell lines we grow in the lab. Which, incidentally, is at the centre of the controversy around HeLa cells. HeLa was the first cell line ever made over 60 years ago and today a PubMed search for “HeLa” return 93274 search results.

Cell lines are an integral part to research in many fields, and these days there are probably thousands of cell lines. Usually, they are generated from patient samples which are immortalised and then can be grown in dishes, put under the microscope, frozen down, thawed and revived, have their DNA sequenced, their protein levels measured, be genetically modified, treated with drugs, and generally make biomedical research possible. As a general rule, work with cancer cell lines is an easy and cheap way to investigate biological concepts, test drugs and validate methods, mainly because cell lines are cheap compared to animal research, readily available, easy to grow, and there are few concerns around ethics and informed consent. This is because although they originate from patients, the cell lines are not considered living beings in the sense that they have feelings and lives and rights; they are for the most part considered research tools. This is an easy argument to make, as almost all cell lines are immortalised and therefore different from the original tissues patients donated, and most importantly they are anonymous, so that any data generated cannot be related back to the person.

But this is exactly what did not happen with HeLa cells. Henrietta Lack’s cells were taken without her knowledge nor consent after she was treated for cervical cancer at Johns Hopkins in 1951. At this point, nobody had managed to grow cells outside the human body, so when Henrietta Lack’s cells started to divide and grow, the researchers were excited, and yet nobody ever told her, or her family. Henrietta Lacks died of her cancer later that year, but her cells survived. For more on this, there is a great Radiolab episode that features interviews with the scientists, as well as Rebecca Skloot and Henrietta Lack’s youngest daughter Deborah Lacks Pullum.

In the 1970s, some researchers did reach out to the Lacks family, not because of ethical concerns or gratitude, but to request blood samples. This naturally led to confusion amongst family members around how Henrietta Lack’s cells could be alive, and be used in labs everywhere, even go to space, while Henrietta herself had been dead for twenty years. Nobody had told them, let alone explained the concept of cell lines to them.

The lack of consent and information are one side, but in addition to being an invaluable research tool, cell lines are also big business: The global market for cell lines development (which includes cell lines and the media they grow in, and other reagents) is worth around 3 billion dollars, and it’s growing fast. There are companies that specialise in making cell lines of certain genotypes that are sold for hundreds of dollars, and different cell types need different growth media and additives in order to grow. This adds a dimension of financial interest, and whether the family should share in the profit derived from research involving HeLa cells.

We have a lot to be grateful for to HeLa cells, and not just biomedical advances. The history of HeLa brought up a plethora of ethical issues around privacy, information, communication and consent that arguably were overdue for discussion. Innovation usually outruns ethics, but while nowadays informed consent is standard for all research involving humans, and patient data is anonymised (or at least pseudonomised and kept confidential), there were no such rules in 1951. There was also apparently no attempt to explain scientific concept and research to non-scientists.

And clearly we still have not fully grasped the issues at hand, as in 2013 researchers sequenced the HeLa cell genome – and published it. Again, without the family’s consent. The main argument in defence of publishing the HeLa genome was that the cell line was too different from the original cells to provide any information on Henrietta Lack’s living relatives. There may some truth in that; cell lines change a lot over time, but even after all these years there will still be information about Henrietta Lack’s and her family in there, and genetic information is still personal and should be kept private.

HeLa cells have gotten around to research labs around the world and even gone to space and on deep sea dives. And they are now even contaminating other cell lines (which could perhaps be interpreted as just karma). Sadly, the spotlight on Henrietta Lack’s life has sparked arguments amongst the family members around the use and distribution of profits and benefits from the book and movie, and the portrayal of Henrietta Lack’s in the story. Johns Hopkins say they have no rights to the cell line, and have not profited from them, and they have established symposiums, scholarships and awards in Henrietta Lack’s honour.

The NIH has established the HeLa Genome Data Access Working Group, which includes members of Henrietta Lack’s family. Any researcher wanting to use the HeLa cell genome in their research has to request the data from this committee, and explain their research plans, and any potential commercialisation. The data may only be used in biomedical research, not ancestry research, and no researcher is allowed to contact the Lacks family directly.

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.


Can we reprogram adult cells into eggs?


By Sophie Balmer, PhD


Oogenesis is the female process necessary to create eggs ready for fertilization. Reproducing these keys steps in culture constitutes a major advance in developmental biology. Last week, a scientific group from Japan amazingly succeeded and published their results in the journal Nature. They replicated the entire cycle of oogenesis in vitro starting from adult skin cells. Upon fertilization of these in vitro eggs and transfer in adult females, they even obtained pups that grew normally to adulthood providing new platforms for the study of developmental biology.


Gamete precursor cells first appear early during embryonic development and are called primordial germ cells. These precursors then migrate to the gonads where they will remodel their genome via two rounds of meiosis to produce either mature oocytes or sperm depending on the sex of the embryo. For oocyte maturation, these two cycles occur at different times: the first one before or shortly after birth and the second one at puberty. The second round of meiosis is incomplete and the oocytes remain blocked in metaphase until fertilization by male gametes. This final event initiates the process of embryonic development, therefore closing the cycle of life.


Up until last week, parts of this life cycle were reproducible in culture. For years, scientists have known how to collect and culture embryos, fertilize them and transfer them to adult females to initiate gestation. This process called in vitro fertilization (IVF) has successfully been applied to humans and has revolutionized the life of millions of individuals suffering specific infertility issues and allowing them to have babies. However only a subset of infertility problems can be solved by IVF.

Additionally, in 2012, the same Japanese group recreated another part of the female gamete development: Dr. Hayashi and colleagues generated mouse primordial germ cells in vitro that once transplanted in female embryos recapitulated oogenesis. Both embryonic stem (ES) cells or induced pluripotent stem (iPS) cells were used for such procedure. ES cells can be derived from embryos before their implantation in the uterus and iPS cells are derived by reprogramming of adult cells. Finally, a couple of months ago, another group also reported being able to transform primordial germ cells collected from mouse embryos into mature oocytes.


However, replicating the full cycle of oogenesis from pluripotent cell lines in a single procedure constitutes an unprecedented discovery. To achieve this, they proceeded in different steps: first, they produced primordial germ cells in vitro from either skin cells (following their de-differentiation into iPS cells) or directly from ES cells. Second, they produced primary oocytes in a specific in vitro environment called “reconstituted ovaries”. Third, they induced maturation of oocyte up until their arrest in meiosis II. This process took approximately the same time as it would take in the female mouse and it is impressive to see how the in vivo and in vitro oocytes are indistinguishable. Of course, this culture system also produced a lot of non-viable eggs and only few make it through the whole process. For example, during the first step of directed differentiation, over half of the oocytes contain chromosome mispairing during meiosis I, which is about 10 times more than in vivo. Additionally, only 30% complete meiosis I as shown by the exclusion of the 1st polar body. However, analysis of other parameters such as the methylation pattern of several genes showed that maternal imprinting was almost complete and that most of the mature oocytes had normal number of chromosomes. Transcription profiling also showed very high similarities between in vitro and in vivo oocytes.

The in vitro oocytes were then used for IVF and transplanted into mouse. Amazingly, some of them developed into pups that were viable, grew up to be fertile and had normal life expectancy without apparent abnormalities. However, the efficiency of such technique is very low as only 3.5% of embryos transplanted were born (compare to over 60% in the case of routine IVF procedures). Embryos that did not go through the end of the pregnancy showed delayed development at various stages, highlighting that there are probably conditions that could be improved for the oocytes to lead to more viable embryos.

Looking at the entire process, the rate of success to obtain eggs ready for transplant is around 7-14% depending on the starting cell line population. Considering how much time these cells spend in culture, this rate seems reasonably good. However, as mentioned above only few develop to birth. Nonetheless, this work constitutes major advancement in the field of developmental biology and will allow researchers to look in greater detail at the entire process of oogenesis and fertilization without worrying about the number of animals needed. We can also expect that, as with every protocol, it will be fine-tuned in the near future. It is already very impressive that the protocol led to viable pups from 6 different cell line populations.


Besides its potential for increasing knowledge in the oogenesis process, the impact of such research might reach beyond the scope of developmental biology. Not surprisingly, these results came with their share of concerns that soon this protocol would be used for humans. How amazing would it be for women who cannot use IVF to use their skin cells and allow them to have babies? Years ago, when IVF was introduced to the world, most people thought that “test-tube” babies were a bad idea. Today, it is used as a routine treatment for infertility problems. However, there is a humongous difference between extracting male and female gametes and engineering them. I do not believe that this protocol will be used on humans any time soon because it requires too many manipulations that we still have no idea how to control. Nonetheless, in theory, this possibility could be attractive. Also, for the most sceptic ones, one of the major reason why this protocol is not adaptable to human right now is that we cannot generate human “reconstituted ovaries”. This step is key for mouse oocytes to grow in vitro and necessitate to collect the gonadal somatic cells in embryos which is impossible in humans. So, until another research group manages to produce somatic gonadal cells from iPS cells, no need to start freaking out 😉



In the Life of a Cell

An introduction to the cell cycle


By Johannes Buheitel, PhD

Omnis cellula e cellula”. We all heard or read this sentence probably sometime during college or grad school and no, it’s not NYU’s university motto. This short Latin phrase, popularized by the German physician/biologist Rudolf Virchow, states a simple fact, which, however, represents a fundamental truth of biology: “All cells come from cells”. It’s so fundamental that we often take it for granted that the basis for all of those really interesting little pathways and mechanisms that we study is life itself; and, moreover, that life is not simply “created” from thin air but can actually only derive from other life. Macroscopically, you (and Elton John) might call this “the circle of life” but microscopically, we’re talking about nothing less than the cell cycle. But what is the cell cycle exactly? What has to happen when and how does the cell maintain this order of events?

The cell cycle’s main purpose is to generate two identical daughter cells from one mother cell by first, duplicating all its genetic content in order to get two copies of each chromosome (DNA replication), and then carefully distributing those two copies into the newly forming daughter cells (mitosis and cytokinesis). These two major chromosomal events take place during S phase (DNA replication) and M phase (mitosis), which during consecutive cycles alternate, separated by two “gap” phases (G1 between M and S phase and G2 between S and M phase; FYI: everything outside M phase is also sometimes also called interphase). It goes without saying that the temporal order of events, G1 to S to G2 to M phase, must be maintained at all times; just imagine trying to divide without previously having replicated your DNA! And not only the order is important, but each phase must also be given enough time to faithfully fulfill its purpose. How is this achieved?

If you want to boil it down, there are two main principles that drive the cell cycle: timely expression and degradation of key proteins and irreversible switch-like transitions, called checkpoints. So let’s try and get an overview over each of these principles.

Overview over the different phases of the cell cycle: G1 (“gap”) phase, S (“synthesis”) phase, G2 phase and M (“mitosis”) phase.

In the early eighties, a scientist called Tim Hunt performed a series of experiments, unknowing that these will turn into a body of work, which will ultimately win him a Nobel prize. For these experiments, he radioactively labeled proteins in sea urchin embryos (yes, you read correctly!) and stumbled across one that exhibited an interesting pattern of abundance over time in that it appeared and vanished in a fashion that was not only cyclic but also seemed to be in sync with the embryos’ division cycles. Dr. Hunt had just found the first member of a protein family, which later turned out to be one of the main drivers of the cell cycle: the cyclins. What do cyclins do? Cyclins are co-activators of cyclin-dependent kinases or Cdks, whose job it is to phosphorylate certain target proteins in order to regulate their function in a cell cycle-dependent fashion. Since Cdks are pretty much around all the time, they need the cyclins to tell them, when to be active and when not to be. There are a variety of different Cdks, which interact very specifically with various cyclins. For example, cyclin D interacts with Cdk4 and 6 to drive the transition from G1 to S phase, while a complex between cyclin B and Cdk1 is required for mitotic entry. This system allows for enough complexity to explain how the proper length of each phase is assured (slow accumulation of a specific cyclin until the respective Cdk can be fully activated), but also how the correct order of events is maintained; because it turns out that the expression of, say, the cyclin assigned to start replication (cyclin E) is dependent on the activity of the Cdk/cyclin complex of the previous phase (in this example: Cdk4/6-cyclin D) via phosphorylation-dependent regulation of transcription factors.

The second principle I was talking about, are checkpoints. A checkpoint is a way for the cell to take a short breath and check if things are running smoothly so far, and if they are not, to halt the cell cycle in order to give itself some time to either resolve the issue or, if that’s not working out, throw in the towel (i.e. apoptosis). Researchers describe more and more checkpoint-like pathways that react to different stimuli all over the cell cycle, but canonically, we distinguish three main ones: the restriction checkpoint at the G1 to S phase transition, the DNA damage checkpoint at the G2 to M phase transition and the spindle assembly checkpoint (SAC) during mitosis at the transition from metaphase to anaphase. What do these checkpoints look for, or in more technical words, what requirements have to be met in order for a checkpoint to become satisfied? The restriction checkpoint integrates a variety of internal and external signals, but is ultimately satisfied by proper activation of S phase Cdk complexes (see above). The DNA damage checkpoint’s main function is to give the cell time to correct DNA damage, which naturally occurs during genome replication but can also be introduced chemically or by ionizing radiation. Therefore, it remains unsatisfied as long as the DNA damage kinases ATM and ATR are active. Finally, the SAC governs one of the most intricate processes of the cell cycle: the formation of the mitotic spindle including proper attachment of each and every chromosome to its microtubules. After a checkpoint becomes satisfied, one or more positive feedback loops spring into action and effectively jump re-start the cell cycle.

As one can imagine, all of these processes must be exquisitely controlled to ensure the mission’s overall success. In future posts, we will explore those mechanisms in more detail and will furthermore discuss, how a handful of biochemical fallacies can have the potential to turn this wonderful circle of life into a wicked cycle of death.

Repair Gone Wrong: Targeting The DNA Damage Response To Treat Cancer

By Gesa Junge, PhD


Our cells are subject to damage every minute of every day, be it from endogenous factors such as reactive oxygen species generated as part of normal cell respiration, or exogenous factors such as UV radiation from the sun. Together, these factors can lead to as many as 60 000 damaged DNA bases per cell per day. Most of these are changes to the DNA bases or single strand breaks (SSBs), which only affect one strand of the double helix, and can usually be repaired before the DNA is replicated and the cell divides. However, about 1% of SSBs escape and become double stand breaks (DSBs) upon DNA replication. DSBs are highly toxic, and a single DSB can be lethal to a cell if not repaired.

Usually, cells are well-equipped to deal with DNA damage and have several pathways that can remove damaged DNA bases and restore the DNA sequence. Nucleotide excision repair (NER, e.g. for UV damage) and base excision repair (BER, for oxidative damage) are the main SSB repair pathways, and homologous recombination (HR) and non-homologous enjoining (NHEJ) repair most DSBs. HR is the more accurate pathways for DSB repair, as it relies on a homologous DNA sequence on the sister chromosome to restore the damaged bases, whereas NHEJ simply relegates the ends of the break, potentially losing genetic information. However, NHEJ can function at any time in the cell cycle whereas HR requires a template and is only active once the DNA is replicated (i.e. in G2 and S-phase).

Depending on the severity of the damage, cells can either stop the cell cycle to allow for repair to take place or, if the damage is too severe, undergo apoptosis and die, which in a multicellular organism is generally favourable to surviving with damaged DNA. If cells are allowed to replicate with unrepaired DNA damage, they pass this damage on to their daughter cells in mitosis, and mutations in the DNA accumulate. While mutations are essential to evolution, they can also be problematic. Genomic instability, and mutations in genes such as those that control the cell cycle and the DNA damage response can increase the risk of developing cancer. For example, germline mutations in ATM, a key protein in HR pathway of DSB repair, leads to Ataxia Telangiectasia (AT), a neurodegenerative disorder. AT sufferers are hypersensitive to DSB-inducing agents such as x-rays, and have a high risk of developing cancer. Deficiencies in NER proteins lead to conditions such as Xeroderma Pigmentosa or Cockayne Syndrome which are characterised by hypersensitivity to UV radiation and an increased risk of skin cancer, and mutations BRCA2, another key HR protein, increase a woman’s risk of developing breast cancer to 60-80% (compared to 13% on average).

Even though deficiencies in DNA repair can predispose to cancer, DNA repair is also emerging as a viable target for cancer therapy. For example, DNA repair inhibitors can be used to sensitise cancer cells to chemotherapy- or radiation-induced damage, making it possible to achieve more tumour cell kill with the same dose of radiation or chemotherapy. However, this approach is not yet used clinically and a major complication is that it often increases both the efficacy as well as the toxicity of treatment.

Another approach is the idea of “synthetic lethality”, which relies on a cancer cell being dependent on a specific DNA repair pathway because it is defective in another, such that deficiency of either one of two pathways is sustainable, but loss of both leads to cell death. This concept was first described by Calvin Bridges in 1922 in a study of fruit flies and is now used in the treatment of breast cancer in the form of an inhibitor of Poly-ADP ribose polymerase (PARP), a key enzyme in the repair of SSBs. Loss of PARP function leads to increased DSBs after cell division due to unrepaired SSBs, which in normal tissue are removed by the DSB repair system. However, BRCA2-deficient tumours are defective in HR and cannot repair the very toxic DSBs, leading to cell death. Therefore, BRCA2-deficient tumours are hypersensitive to PARP inhibitors, which are now an approved therapy for advanced BRCA2-deficient breast and ovarian cancer.

PARP inhibitors are a good example of a so-called “target therapy” for cancer, which is the concept of targeting the molecular characteristics that distinguish the tumour cell from healthy cells (in this case, BRCA2 deficiency), as opposed to most older, cytotoxic chemotherapies, which generally target rapidly dividing cells by inducing DNA damage, and can actually lead to second cancers. With an improved understanding of the molecular differences between normal and tumour cells, cancer therapy is slowly moving away from non-specific cytotoxic drugs towards more tolerable and effective treatments.



By Sophie Balmer, PhD

One of the first questions that comes to my mind when discussing the emergence of cancer cells is how my immune system recognizes that my own cells have been transformed? This process is commonly termed cancer immunosurveillance. In the prevalent model, the adaptive immune system composed of lymphocytes circulating in the blood stream plays the main function. However, recent findings describe specific immune cells already present within the tissue, a.k.a. tissue-resident lymphocytes, and how they trigger the first immune response against cancer cells, allowing a much faster reaction in an attempt to eradicate transformed cells.


The cancer immunosurveillance concept hypothesizes that sentinel thymus-derived immune cells constantly survey tissues for the presence of nascent transformed cells. Cancer immunosurveillance was first suggested in the early 1900’s by Dr. Erlich but it took another fifty years for Dr. Thomas and Dr. Burnet to revisit this model and speculate about the presence of transformed cells induced inflammation and antigen-specific lymphocyte responses. Additionally, Dr. Prehn and Dr. Main estimated that chemically-induced tumor triggered the synthesis of antigen at the surface of cancerous cells that could be recognized by the immune system. Countless studies arose from these hypotheses and either validated or disproved these models. The latest attempt was published a little over a month ago, in a paper by Dr. Dadi and colleagues, describing a new mechanism for the immune system to respond to nascent cancer lesions by activating specific resident lymphocytes.


In this study, the authors used a genetically-induced tumor model (the MMTV-PyMT spontaneous mammary cancer mouse model) to analyze the in vivo response of the immune system to nascent transformed cells. Most studies have been performed using chemically-induced tumors or tumor transplantation into a healthy host but these do not account for the initial environment of the nascent tumor. The spontaneous model the authors use rapidly exhibits developing cancer lesion (in 8-week old mice), allowing the analysis of cellular populations present near transformed cells.


To analyze which immune cell types are present near cancer lesions, the authors performed several analyses. First, they measure the levels of granzyme B, a serine protease found in granules synthesized by cytotoxic lymphocytes to generate apoptosis of targeted cells, and show that PyMT mice have elevated levels of granzyme B when compared to wild-type mouse. Moreover, similar analysis of PyMT secondary lymphoid organs show that this response was restricted to the transformed tissue.

During the first steps of immune responses, conventional natural killer (cNK) cells as well as innate lymphoid cells (ILC) are found in tumor microenvironments. In this model however, sorting of cells located in the vicinity of the lesion identified unconventional populations of immune cells, derived from innate, TCRab and TCRgd lineages. Indeed, their RNA-seq profiling reveal a specific gene signature characterized by high expression of the NK receptor NK1.1 but also the integrins CD49a and CD103. As these newly identified cells share part of their transcriptome with type 1 ILCs, the authors named them type 1-like ILCs (ILC1ls) and type 1 innate-like T cells (ILTC1s). In addition, transcripts encoding several immune effectors as well as apoptosis-inducing factors are upregulated in these cells, likely indicating that they trigger several pathways to eliminate transformed cells.

The authors also suggest that cNK cells are not required for immunosurveillance in this model and the unconventional lymphocytes described in this paper are regulated by the interleukine-15 (IL-15) in a dose-dependent way. Mice overexpressing IL-15 exhibit higher proliferation of these resident lymphocytes and tumor regression. Secretion of IL-15 in the tumor microenvironment might therefore promote cancer immunosurveillance.


In contradiction with the conventional view that recirculating populations of immune cells survey tissues for cellular transformation, ILC1ls and ILTC1s are tissue-resident lymphocytes. Their gene signature indicates that transcripts encoding motility-related genes are downregulated in these cells. Moreover, parabiosis experiments, during which two congenically marked mice are surgically united and share their blood stream, are performed to determine whether they are resident or circulating cells. The amounts of non-host ILC1s and ILTC1s are much reduced when compared to other recirculating immune cell type demonstrating that these cells are tissue-resident lymphocytes. Single-cell killing assays also determine that ILC1ls and ILTC1s are highly efficient at inducing apoptosis of tumor cells, which is more likely dependent on the lytic granules pathway.


Although the cancer immunosurveillance concept has been around for decades, it is still highly debated. Overall, these results shed light on this confusing field and bring up several questions. The signals recognized by this immune response are still unknown. Although the authors suggest that IL-15 might regulate the proliferation and/or activation of these cells, the source of IL-15 remains to be found. In addition, these cells might promote cancer immunosurveillance but are not sufficient to eradicate tumor cells and determining the cascade of signals induced by these resident lymphocytes will be required to ascertain their role. Establishing the limit of their efficiency as well as the mechanisms activated by transformed cell to escape their surveillance will also be crucial. Finally, one of the most important question to consider is how one could manipulate the activity of tissue-resident lymphocytes in cancer immunotherapy.

Epigenetic Inheritance, Trauma and the Holocaust


By Alison Bernstein, PhD

Since my research interests focus on environmental impacts on health and how epigenetic processes mediate those effects, my mother sent me this article, “Study of Holocaust survivors finds trauma passed on to children’s genes“, from The Guardian. This article reports the recent paper, “Holocaust exposure induced intergenerational effects on FKBP5 methylation“, in Biological Psychiatry. I get overly excited by teachable moments so I decided to take the opportunity to teach some more epigenetics (see my pages on Facebook or Google+ for my Intro to Epigenetics series).

Epigenetics literally means “over the genome”. It encompasses all meiotically and mitotically heritable changes in gene expression that are not coded in the DNA sequence itself. If we break that down, there are some key points to note:

  • “Not coded in the DNA”: There is no change in the DNA sequence. Thus, for these to be heritable, there must be mechanisms of inheritance besides DNA replication.
  • “Changes in gene expression”: The underlying assumption of all epigenetic studies should be that these changes alter gene expression (or change how inducible or repressible gene expression is, but that’s harder to measure).
  • “Meiotically and mitotically heritable”: This means heritable through cell division, but not necessarily heritable from parent to offspring.

Epigenetics generally refers to 4 mechanisms: DNA methylation (and other modifications to cytosine), histone modifications, non-coding RNAs, and long-range chromatin interactions (3D structure of chromosomes). In this paper, the authors focused on DNA methylation and identified changes in DNA methylation that occur in people who were in a Nazi concentration camp, witnessed or experienced torture, or hid from the Nazis during World War II. Similar changes were seen in their children. This transmission of a trait from parents to children is called intergenerational inheritance.

The effects of severe stress and other exposures has been shown to be inherited intergenerationally, multigenerationally (to grandchildren) and sometimes even transgenerationally (to great-grandchildren), both in animals and in people. The Dutch famine of 1944 and the polybrominated biphenyl exposure in Michigan in 1978 have provided evidence that exposures that occur prior to conception and in utero can have lasting effects on subsequent generations. However, it is difficult to separate out the different mechanisms that contribute to the inheritance of traits to subsequent generations. Thus, it is an important research question to ask how the effects of trauma, stress and other exposures are passed from generation to generation. This is the question the scientists wanted to address in this paper: is there an epigenetic component to the intergenerational inheritance of the effects of trauma?

Epigenetic Inheritance

This paper provides direct evidence in humans that the epigenetic effects of pre-conception stress can be seen in both parents and offspring. The authors looked at one specific gene only – FKBP5 – because it is known to be involved in the response to high glucocorticoid levels (a biological signal for stress) and is a possible novel target for antidepressant medication. They looked for changes in DNA methylation in glucocorticoid response elements within this gene. Response elements are sequences of DNA that bind to specific transcription factors and regulate transcription of genes. In this case, glucocorticoid response elements are bound by glucocorticoid hormones and their receptors to regulate expression of the gene containing the response element. They found changes in DNA methylation in these specific elements of the specific FKBP5 gene in Jewish Holocaust survivors and their children, but not in other Jewish people of similar age. This observed change in DNA methylation of the FKBP5 gene was in the opposite direction in parents and offspring, yet we do not yet have an explanation as to why this change would be different in parents and offspring. Thus, it is actually impossible to say from the results of this paper if these epigenetic changes are due to direct effects of stress and high glucocorticoid levels (or other shared environmental factors) or to inheritance of epigenetic marks.

Let’s say a woman or girl lived through the Holocaust. She and her eggs were exposed to high glucocorticoid levels, and other effects, due to stress. If a woman was pregnant during this time, she, her eggs and her in utero daughters’ eggs were exposed. So that’s 2, and possibly, 3 generations directly exposed to the stress. Until you get to the 4th generation, there is still a possibility of direct exposure. It might be epigenetic, but it is also possible that it’s still a result of direct exposure. Changes must be observed in the generation the great-grandchildren to definitively say that they are epigenetically inherited and not a result of direct exposure. In general, the great-grandchildren are the first generation that was definitely not directly exposed to the stressor. However, in this case, they looked at preconception stress, so looking at the 3rd generation (grandchildren) would be sufficient to differentiate between epigenetic inheritance and direct exposure.

This paper only looks at parents and their children. So the eggs that produced ALL those children were directly exposed (since females are born with all their eggs) to the trauma. It’s possible that high glucocorticoid levels directly affect the methylation of FKBP5 in the eggs as well in cells of the parent. The discussion of the paper itself goes into this, but the article overlooked this point and it’s a really important point to understand if you are interested in epigenetic inheritance.

From the discussion section of the paper:

“The main finding in this study is that Holocaust survivors and their offspring have methylation changes on the same site in a functional intronic region of the FKBP5 gene, a GR binding sequence in intron 7, but in the opposite direction. To our knowledge, these results provide the first demonstration of transmission of preconception stress effects resulting in epigenetic changes in both exposed parents and their offspring in adult humans. Bin 3/site 6 methylation was not associated with the FKBP5 risk-allele, and could not be attributed to the offspring’s own trauma exposure, their own psychopathology, or other examined characteristics that might independently affect methylation of this gene. Yet, it could be attributed to Holocaust exposure in the F0.

It is not possible to infer mechanisms of transmission from these data. It was not possible to disentangle the influence of parental gender, including in utero effects, since both Holocaust parents were survivors. Epigenetic effects in maternal or paternal gametes are a potential explanation for epigenetic effects in offspring, but blood samples will not permit ascertainment of gamete dependent transmission. What can be detected in blood samples is parental and offspring experience-dependent epigenetic modifications. Future prospective, longitudinal studies of high risk trauma survivors prior to conception, during pregnancy and postpartum may uncover sources of epigenetic influences.”

The paper reports evidence that the epigenetic effects of stress and trauma can be seen in both parents and offspring. However, there are a lot of variables that may cause similar epigenetic changes in parents and offspring. Further studies are needed to really know what the mechanism of these shared epigenetic marks are, before we can confidently assert that the epigenetic changes observed in parents and offspring are due to epigenetic inheritance. As with all good science, this paper answers a question while, at the same time, raising additional questions for future research.

This article was originally published on The Sound of Science blog in August 2015.

Leaving Your Mark on the World

By Danielle Gerhard


The idea that transgenerational inheritance of salient life experiences exists has only recently entered the world of experimental research. French scientist Jean-Baptiste Lamarck proposed the idea that acquired traits throughout an organism’s life could be passed along to offspring. This theory of inheritance was originally disregarded in favor of Mendelian genetics, or the inheritance of phenotypic traits isn’t a blending of the traits but instead a specific combination of alleles to form a unique gene encoding the phenotypic trait. However, inheritance is much more complicated than either theory allows for. While Lamarckian inheritance has largely been negated by modern genetics, recent findings in the field of genetics have caused some to revisit l’influence des circonstances, or, the influence of circumstances.


Over the past decade, efforts have shifted towards understanding the mechanisms underlying the non-Mendelian inheritance of experience-dependent information. While still conserving most of the rules of Mendelian inheritance, new discoveries like epigenetics and prions challenge the central dogma of molecular biology. Epigenetics is the study of heritable changes in gene activity as a result of environmental factors. These changes do not affect DNA sequences directly but instead impact processes that regulate gene activity such as DNA methylation and histone acetylation.


Epigenetics has transformed how psychologists approach understanding the development of psychological disorders. The first study to report epigenetic effects on behavior came from the lab of Michael Meany and Moshe Szyf at McGill University in the early 2000s. In a 2004 Nature Neuroscience paper they report differential DNA methylation in pups raised by high licking and grooming mothers compared to pups raised by low licking and grooming mother. Following these initial findings, neuroscientists have begun using epigenetic techniques to better understand how parental life experiences, such as stress and depression, can shape the epigenome of their offspring.


Recent research coming out from the lab of Tracy Bale of the University of Pennsylvania has investigated the heritability of behavioral phenotypes. A 2013 Journal of Neuroscience paper found that stressed males went on to produce offspring with blunted hypothalamic pituitary (HPA) axis responsivity. In simpler terms, when the offspring were presented with a brief, stressful event they had a reduction in the production of the stress hormone corticosterone (cortisol in humans), symptomatic of a predisposition to psychopathology. In contrast, an adaptive response to acute stressors is a transient increase in corticosterone that signals a negative feedback loops to subsequently silence the stress response.


The other key finding from this prior study is the identification of nine small non-coding RNA sperm microRNAs (miRs) increased in stressed sires. These findings begin to delve into how paternal experience can influence germ cell transmission but does not explain how selective increases in these sperm miRs might effect oocyte development in order to cause the observed phenotypic and hormonal deficits seen in adult offspring.


A recent study from the lab published in PNAS builds off of these initial findings to further investigate the mechanisms underlying transgenerational effects of paternal stress. Using the previously identified nine sperm miRs, the researchers performed a multi-miR injection into single-cell mouse zygotes that were introduced into healthy surrogate females. To confirm that all nine of the sperm miRs were required to recapitulate the stress phenotype, another set of single-cell mouse zygotes were microinjected with a single sperm miR. Furthermore, a final set of zygotes received none of the sperm miRs. Following a normal rearing schedule, the adult offspring were briefly exposed to an acute stressor and blood was collected to analyze changes in stress hormones. As hypothesized, male and female adult offspring from the multi-miR group had a blunted stress response relative to both controls.


To further investigate potential effects on neural development, the researchers dissected out the paraventricular nucleus (PVN) of the hypothalamus, a region of the brain that has been previously identified by the group to be involved in regulation of the stress response. Using RNA sequencing and gene set enrichment analysis (GSEA) techniques they found a decrease in genes involved in collagen formation and extracellular matrix organization which the authors go on to hypothesize could be modifying cerebral circulation and blood brain barrier integrity.


The final experiment in the study examined the postfertilization effects of multi-miR injected zygotes. Specifically, the investigators were interested in the direct, combined effect of the nine identified sperm miRs on stored maternal mRNA. Using a similar design as the initial experiment, the zygote mRNA was collected and amplified 24 hours after miR injection in order to examine differential gene expression. The researchers found that microinjection of the nine sperm miRs reduced stored maternal mRNA of candidate genes.


This study is significant as it has never been shown that paternally derived miRs play a regulatory role in zygote miR degradation. In simpler terms, these findings contradict the conventional belief that zygote development is solely maternally driven. Paternal models of transgenerational inheritance of salient life experiences are useful as they avoid confounding maternal influences in development. Studies investigating the effects of paternal drug use, malnutrition, and psychopathology are ongoing.


Not only do early life experiences influence the epigenome passed down to offspring but recent work out of the University of Copenhagen suggests that our diet may also have long-lasting, transgenerational effects. A study that will be published in Cell Metabolism next year examined the effects of obesity on the epigenome. They report differential small non-coding RNA expression and DNA methylation of genes involved in central nervous system development in the spermatozoa of obese men compared to lean controls. Before you start feeling guilty about the 15 jelly donuts you ate this morning, there is hope that epigenetics can also work in our favor. The authors present data on obese men who have undergone bariatric surgery-induced weight loss and they show a remodeling of DNA methylation in spermatozoa.


Although still a nascent field, epigenetics has promise for better understanding intergenerational transmission of risk to developing a psychopathology or disease. The ultimate goal of treatment is to identify patterns of epigenetic alternations across susceptible or diagnosed individuals and develop agents that aim to modify epigenetic processes responsible for regulating genes of interest. I would argue that it will one day be necessary for epigenetics and pharmacogenetics, another burgeoning field, to come into cahoots with one another to not only identify the epigenetic markers of a disease but to identify the markers on an person by person basis. However, because the fields of epigenetics and pharmacogenetics are still in the early stages, the tools and techniques currently available limit them. As a result, researchers are able to extract correlations in many of their studies but unable to determine potential causality. Therefore, longitudinal, transgenerational studies like those from the labs of Tracy Bale and others are necessary to provide insight into the lability of our epigenome in response to lifelong experiences.

Development On the Fly: An Interview with Dr. Thomas Gregor

By John McLaughlin


Thomas Gregor is a biophysicist and Professor at Princeton University. His Laboratory for the Physics of Life uses both Drosophila melanogaster and Dictyostelium discoideum as model systems to understand developmental processes from a physical perspective.


Could you briefly describe your educational path from undergraduate to faculty member at Princeton?

TG: As an undergraduate, I studied physics in Geneva, and then moved into theoretical physics and math. I came to Princeton, initially for a theoretical physics PhD; I switched during my time here to theoretical biophysics and then realized that it makes sense to combine this with experiments. I ended up doing a PhD between three complementary disciplines. My main advisor was Bill Bialek, a theoretical physicist. My other two were David Tank, an experimental neuroscientist, and Eric Wieschaus, a fly geneticist. So I had both experiment and theory, from a biological and a physical side. I then went to Tokyo for a brief post-doc, during which I continued in that interface. But I changed model organisms: I switched from a multicellular, embryonic system to looking at populations of single cells [the social amoeba Dictyostelium discoideum]. As a physicist you’re not married to model organisms. When I came back to start my lab at Princeton in 2009, I kept both the fly and the amoeba systems.


What is the overall goal of your lab’s research program?

TG: Basically, to find physical principles behind biological phenomena. How can we come up with a larger, principled understanding that goes beyond the molecular details of any one particular system? I mostly look at genetic networks and try to understand their global properties.


Do you think the approaches of biologists and physicists are very different, and if so are they complementary?

TG: I’m driven by the physical aspects of things, but I’m also realistic enough to see what can now be done in biological systems, in terms of data collection and what we can test. To find the overlap between them is kind of an art, and I think that’s where I’m trying to come in.


Do you have any scientific role models who have shaped how you approach science?

TG: The three that I mentioned: Bialek influenced me in the types of questions that speak to me; Tank had a very thorough experimental approach that taught me how to make real, physics-style measurements; and Wieschaus brought a lot of enthusiasm and knowledge of the system.


Your lab has been studying developmental reproducibility and precision, in the patterning of the fly Drosophila melanogaster. In a 2014 paper1, you showed that levels of the anterior determinant bicoid mRNA vary by only ~9% between different embryos. This is a very similar value to the ~10% variation in Bicoid protein levels between embryos, which you demonstrated several years earlier2. So it seems that this reproducibility occurs even at the mRNA level.

TG: Before going into this, the general thought in the field is that things were very noisy initially, and as the developmental path goes along it becomes more refined and things become more precise. This paper basically asked whether the precision is inherited from the mother, or the embryo needs to acquire it. Because the fluctuations in mRNA, from the mother, completely mimic the fluctuations in protein that the zygote expresses, that told us that the mother lays the groundwork, and passes on a very reproducible pattern. So there’s no necessity for a mechanism that reduces fluctuations from the mRNA to the protein level.


Continuing on the theme of precision: in a separate paper from the same year3, your lab showed that the wing structure among different adult flies is identical to within less than a single cell width. Did you have any prior expectations going into this study, and did the results surprise you?

TG: Before looking at the wing, I had kind of made up my mind. I had first seen single cell precision in patterning of gene expression boundaries in the embryo. But I also knew that it’s always better to make a measurement first, and it seems that things are much more precise and reproducible in biology than we think, given the idea of “sloppiness” that we have.


Do you think that a high level of reproducibility is a general feature of development, or varies widely among different types of species?

TG: It’s a philosophical question in a way, because I haven’t looked. I think what we found in the embryo is not special to the fly; specific mechanisms for getting there might be unique to the fly. For instance, we have also shown in a recent paper from 2013 that transcription is just as noisy in flies as it is in bacteria, hugely noisy. So, physical mechanisms like temporal and spatial averaging seem enough to reduce the high ubiquitous noise that transcription has to the very fine, reproducible patterns that you see in the fly. The specific mechanisms that reduce noise will be very different from species to species, but I think overall the fact that development is precise and reproducible is something we may one day be able to call a principle.


If you could make any changes to scientific institutions, such as the current funding system, journal peer review, etc. what would they be?

TG: One thing that might be nice is if we didn’t have to fund graduate students for the first five years of their career; it would be nice to have more streamlined training grants, not only for U.S. but also international graduate students. And so, graduate students wouldn’t have to worry. They should be free to choose a school based on their scientific interests.

For peer review in journals, the problem is the sheer volume of output is becoming so high. One way to keep a peer review system, is either to pay the reviewers money, or to put everything on the bioRxiv [bio archive is a pre-print server for the life sciences] and let some other means determine how to evaluate a paper. I don’t read papers from looking at the top journals’ table of contents every week, I read them because I see people talk about it on Twitter, or my colleagues tell me I should look at that paper, or because I hear about the work in a talk and decide to see what else the guy is doing.

A lot of people are advocating the new metrics – citations, citation rates, H-index – which are so dependent on the particular field and not necessarily a good measure of impact. In 100 years, are we going to look more at those papers than the ones that currently get very few citations? We don’t know. I don’t think the solution is out there yet.


Do you have any advice for young scientists – current PhD students or post-doctoral fellows – for being successful in science?

TG: My advice would be to focus on one very impactful finding. If it’s very thorough and good science, it will be seen. Also, nothing comes from nothing. You need to put in the hours if you want to get a job in academia. And I think that’s one of the ways to measure a good scientist, because knowledge in experimental science comes from new, good data.

What are some future goals of your lab’s research?

TG: We’ve been looking at the genetic network in the fly embryo, trying to understand properties of that network. Medium term, we want to incorporate a slightly different angle, which is looking at the link between transcriptional regulation and the 3D architecture of the genome. In the living embryo, we want to look at how individual pieces of DNA interact, and how that influences transcription and eventually patterning. In the longer term, I don’t know yet; I just got tenure, so I need to sit back. Everything is open. That is what’s nice about being a physicist; you’re not married to your biological past so much.


In your opinion, what are the most exciting developments happening in biology right now, whether in your own field or elsewhere?

TG: It’s definitely the fact that so many different disciplines have stormed into biology, making it a very multidisciplinary science. I think it makes the life sciences a very vibrant, communal enterprise. Hopefully the next decades will show the fruits of those interactions.


This question is asked very often: How do you balance your lab and family life?

TG: When you start thinking about having a family in science, things become much more complicated. Since I’ve had children, my workload went down a lot. My wife is also a scientist, and for her it’s much harder because she’s not yet tenured. As much as people look at the CV and see how many high-profile papers you have, they should also look at it and see your family and life situation. And for women in science, despite all the efforts that have been made, I don’t think we’re there yet.



[ordered_list style=”decimal”]

  1. Petkova, MD et al. Maternal origins of developmental reproducibility. Current Biology. 2014. 24(11).
  2. Gregor, T et al. Probing the limits to positional information. Cell. 2007. 130(1).
  3. Abouchar, L et al. Fly wing vein patterns have spatial reproducibility of a single cell. J R Soc Interface. 2014. 11(97).