Information processing in neural and gene regulatory networks

Speaker: Gašper Tkačik
Department:
IST Austria
Subject:
Information processing in neural and gene regulatory networks
Location: A1.100 TU Delft
Date: 22-03-2017

Author: Kristian Blom

On the 22nd of March I visited a seminar given by Gašper Tkačik, a theoretical physicist who is interested in using statistical physics and information theory to explain phenomena related to the cell. The most fundamental principle that underlies all the research that dr. Tkačik conducts is that information processing networks have evolved or adapted to maximize the information transmitted from their inputs to the outputs, given the biophysical noise and resource constraints.

Dr. Tkačik showed us multiple examples of his research during his talk. For now I’d like to focus on the most interesting one (from my point of view), which is about reading the positional code in early development. It is commonly known that a morphogen gradient in early development generates different cell types in distinct spatial orders. This is called the French flag model. Despite decades of biological study, a quantitative answer to how much appositional information there is in an expression pattern remained unanswered. Therefore Dr. Tkačik to look at the French flag model from an information theory point of view and asked the following question: How much information is there in spatial patterns of gene expression? Using the gap genes in the Drosophila embryo he measured the amount of information in bits. I will now discuss shortly how one can measure the information contained in gap genes.

Figure 1: Normalized dorsal profiles of fluorescence intensity, which we identify as Hb expression level g, from 24 embryos selected in a 38- to 48-min time interval after the beginning of nuclear cycle 14. Considering all points with g = 0.1, 0.5, or 0.9 (Left) , yields conditional distributions with probability densities P(x|g) (Right). Note that these distributions are much more sharply concentrated than the uniform distribution P(x) shown in light gray. Image adapted from: Dubuis, J.O.; Tkačik, G.; Wieschaus, E.F.; Gregor, T; Bialek, W. PNAS, 2013, 110 (41), pp 16301-16308

We start by looking at the early stages of Drosphila development. At this stage most cells are similar in morphology, so we do not have any information about the position of cell when we neglect gene expression information. Mathematically we can say that the position of the cell is drawn from a distribution of possibilities P(x). If we know take into account the gene expression levels, our uncertainty in position is reduced.  Looking specifically at the expression levels of the hunchback gap (Hb) gene (figure 1), one can see that a certain expression level (g) is not a unique indicator for the position of the cell along the posterior/anterior axis.  Instead there is a range of positions that have the value g. Let P(x|g) be the conditional probability that a cell with expression level g is located at position x.

We define the entropy  of our two probability distributions as:

The information gain due to an observation of the hg expression level at on cell is now given by

From this point I will leave the mathematical expressions as it is, but I challenge you to get a firm understanding of why the final expression represents the information gain. After a small adaption to the final formula, Dr. Tkačik  used that result to make a ‘’direct’’ measurement of the amount of information carried in the gap genes. Using this method he found that individual genes carry almost two bits of information. In the extension of this result he also found that four gap genes carry enough information to define a cell’s location within an error bar of ~1% along the anterior/posterior axis of the embryo. How cool is that!

Although the talk went a bit fast, the content was really good. During the talk I was reminded of the lectures we had during evolutionary & developmental biology (evodevo), since it was this course where I got familiar with the gap genes in drosophila development. Therefore I decided to inform one of the evodevo teachers with the content of this talk, because it might be of good use in the future for them. Although it sounds a bit cliché, afterwards I was again (it happens on a regular basis) astonished by the fact that nanobiology is a really strong field of science. What Dr. Tkačik did fits very well into our program because he used mathematics, especially information theory, to understand why those gap genes function the way they do. For me it was really a wakeup call to keep questioning myself: Why? If one keeps asking this again and again, I think at some point you will find yourself in the fields of mathematics and physics where the answer will be waiting for you to be found.

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Evolution and Assembly of Eukaryotic Chromatin

Speaker: Fransesca Mattiroli

Department: Lugi Lab, University of Colorado Boulder

Subject: Evolution and assembly of eukaryotic Chromatin

Location: TU Delft, Bionanoscience department

Date: 10-02-2017

Author: Mirte Golverdingen

 

Fransesca Mattiroli’s research is focussed on the DNA packaging units called nucleosomes. These structures organize DNA in the eukaryotic cell nucleus. Nucleosomes are formed by an octameric complex of folded histone dimers called the H3-H4 and H2A-H2B dimers. In mammals, the histones have histone tails which highly contribute to post-translational modifications and they stabilize the nucleosome. Nucleosomes need to assemble and disassemble when they bind to the genome DNA. Histone modifications and variants are dynamic and can promote or inhibit certain interactions. The nucleosome dynamics and compositions have a direct effect on transcription, translation and repair.

The first main interest of Mattiroli is the evolutionary origin of the nucleosome. The nucleosomes are very well conserved through species. Mattiroli focusses on the structural conservation of the histone dimers in Archaea. They, however, miss the tails that contribute to post-translational modification. So, how do these species organize their archaeal genome?

The archaeal histone binding to DNA is similar to eukaryotic histone binding. Archael histones, however, do not form octamers. They can form a much longer structure instead, called nucleosomal ramps. In Vivo, this structure also forms, the longest ramp they found was 90 bp long. So, they found a new way of arranging histone DNA complexes.

Histones are formed on the DNA in two steps, first, two H3-H4 dimers form a tetrasome, then two H2A-H2B dimers attach to this tetrasome forming a nucleosome. Histone chaperones shield the charges of the histones and facilitate their deposition on DNA. However, not much is known on how the chaperones actually contribute to this deposition step. The Chromatin Assembly Factor 1, CAF-1, is Mattiroli’s main interest. CAF-1 mediates in this histone deposition step and is essential in multicellular organisms. Matteroli tried to understand how CAF-1 contributes to the deposition step.

Mattiroli’s first step was to research how CAF-1 binds the H3-H4 dimer. She used mass spectrometry (HX-MS) with a hydrogen-deuterium exchange. She could, in this way, measure the change in mass and which regions have the largest changes in deuterium uptake. This region could then be the binding site of CAF-1 on H3-H4. When CAF-1 binds to the dimer, they see a stabilization of the dimer. This result indicates the following hypothesis: Only if a H3-H4 dimer is bound to CAF-1 it can form a tetrasome.

A next step for Mattiroli was to test if CAF-1 can form nucleosomes in vitro, in absence of other proteins. To test this, Mattiroli mixed CAF-1, histones and DNA, treat them with micrcococcal nuclease to digest unprotected DNA and purified and quantified the length of DNA covered by histones. The result showed that CAF-1 is able to assemble tetrasomes, and therefore enabling nucleosome formation in vitro.

So, how is the H3-H4 tetrasome on the DNA formed? Mattiroli used increased lengths of DNA, to trap any intermediates in the process. Mattiroli showed that the forming of the H3-H4 dimer activates the DNA binding of the dimer. The key intermediate that mediates the DNA binding results to be two CAF-1 units. This was the most interesting result so far, because it was never showed before that two independent CAF-1 were involved in the H3-H4 DNA binding.

The interesting and clear seminar showed again how complex the system of DNA and all the DNA-interacting molecules is. The research of Mattiroli gives a good foundation for more research to nucleosomes and their interaction with DNA. Bringing us closer to fully understand the biological system of DNA.

Honours 7

Figure 1. Canonical and variant nucleosomes

(A) Elements of the histone fold and structures of Xenopus leavis H2A–H2B, H3–H4 and (H3–H4)2 (PDB ID: 1KX5). (B) Structure of the canonical Xenopus leavis nucleosome (PDB ID: 1KX5). Other nucleosome structures, such as the human nucleosome, are structurally similar. (C) Structure of the CenH3CENP‐A‐containing nucleosome (PDB ID: 3AN2). (D) Zoomed view of the αN helix of CenH3CENP‐A (left) and H3 (right) involved in stabilizing the DNA ends. Histone H3 is blue, CenH3CENP‐A is cyan, H4 is green, H2A is yellow, H2B is red, and DNA is white.

Adapted From: Mattiroli, F., D’Arcy, S., & Luger, K. (2015). The right place at the right time: chaperoning core histone variants. EMBO reports, e201540840.

Nanotechnology for biophysics: from single molecules towards synthetic cells

Speaker: Cees Dekker
Department:
Bionanoscience
Subject:
Nanotechnology for biophysics: from single molecules towards synthetic cells
Location: Lecture room E (TU Delft)
Date: 09-02-2017

Kristian Blom

After six months of following theoretical physics and astronomy courses for my minor, I was looking forward to hear something about nanobiology again. Therefore I took the opportunity to visit a seminar given by Cees Dekker for the quantum nanoscience department about nanotech nology for biophysics.

To break the ice between the applied physicists (audience) and the nanobiologist,  prof. Dekker started his talk with a quick review of his scientific career. In 1984 he started his career in solid state physics (which impressed the audience for sure) and over the years he got more and more fascinated by biophysics and nanobiology. Therefore he made the decision to switch from quantum physics to biophysics around 2000. In the past 15 years Prof. Dekker mainly focused on the study of cellular components using the top-down approach. For this kind of research nanotechniques are used to probe the biological cell at its most fundamental level; single biomolecules.

So what is nanotechnology? Specifically in the field of biophysics we can describe nanotechnology as a toolbox of instruments for studies at the single-atom and single-molecule level. A very famous example of this is the scanning tunneling microscope. Another example are the optical and magnetic tweezer with which you can measure the mechanical properties of DNA. Prof. Dekker explained the audience that with a magnetic tweezer you can coil up a DNA strand by applying an external rotating magnetic field to a bead with one end of a DNA molecule is attached. 

Currently the research group of prof. Dekker focuses on three main topics: Single-molecule biophysics of DNA and DNA-protein complexes, solid-state nanopores and its applications, and finally bacteria in nanostructures. I will discuss the former topic in a bit more detail by explaining one of the recent findings of  the Dekker group in this field.

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Figure 1 – Intercalation-induced Supercoiling of DNA (ISD) Source: Ganji, M.; Hyun Kim, S.; van der Torre, J.; Abbondanzieri, E.; Dekker, C. Nano Lett, 2016, 16 (7), pp 4699-4707

The subject of this recent finding is on DNA supercoiling, which is the over- (positively coiled) or under-winding (negatively coiled) of the DNA strand. Since supercoiling plays a very important role in biological processes such as DNA compaction, gene expression and DNA replication, it is very important for the cell to keep control of the supercoiling state of DNA. A specific kind of supercoiled DNA configuration is the plectoneme; a DNA helix which is coiled onto itself. By using an intercalating dye the Dekker group induced supercoils within a linear DNA molecule that was bound to a surface at its two ends. Thereafter they investigated the plectoneme position and dynamics using epifluorescence microscopy. What they found is that the observed plectoneme density and the nucleation and termination rates showed position-dependent variations. More specifically, they found a strong peak in the plectoneme density, nucleation and termination rate near one end of the DNA (see figure 2). As nucleation of a plectoneme requires a lot of bending energy, a locally region where the DNA is already bend a little bit (intrinsic curvature) by external structures (e.g. DNA-bound proteins) would significantly promote the formation and growth of a plectoneme at this site. Therefore the Dekker group inserted a 10-nucleotide mismatched sequence in the middle of the DNA strand. They observed that the plectoneme density showed a peak at the position of the mismatched sequence. So, as expected, flexible DNA bubbles promote the plectoneme formation.

dna-sequence-dependent-pinning-of-plectonemes
Figure 2 – DNA sequence-dependent pinning of plectonemes. (A) Plectoneme densities of 46 identical DNA molecules (thin lines) and their average (red thick line). (B) Averaged nucleation (orange) and termination (blue) rates observed. C) Averaged position-dependent plectoneme size distributions. Source: Ganji, M.; Hyun Kim, S.; van der Torre, J.; Abbondanzieri, E.; Dekker, C. Nano Lett, 2016, 16 (7), pp 4699-4707

My heart leapt for joy when prof. Dekker mentioned that a theoretical model will be made about the nucleation and termination of plectonemes. Although experimental data tells us more about the truth (if the experiment is done properly) than a theoretical model, I think you can only understand a physical phenomenon in the very detail when someone can explain it using the fundamental laws of nature. After this very interesting part of the talk prof. Dekker quickly went over the other two topics where is research group is interested in. At the very end someone from the audience asked if quantum mechanics plays a significant role in the functioning of a cell. The answer of prof. Dekker was that it isn’t that important for phenomenon in the cell (except for photosynthesis and energy conversion). This disappointed me a little bit, since one of my future plans is to use quantum mechanics to get a better understanding of the cell (therefore I will also follow quantum mechanics in the coming semester). But luckily not everything is discovered so far, so for me there is still hope that one day quantum mechanics will be an essential part of every cell biology course!

Recent insights in the pathophysiology of chronic myelomonocytic leukemia

                                                                                                                                                    Aïsha Mientjes

                                                                                                                             aishamientjes@gmail.com

                                                                                                                                                                             4460960                       

Speaker: Eric Solary       

Department: Hematology

Subject: Recent insights in the pathophysiology of chronic myelomonocytic leukemia. 

Location: Erasmus MC  

Date: 19-12-2016           

The presentation was about CMML (chronic myelomonocytic leukemia), the genetic background of this disease and the treatments. The speaker begun by explaining a little bit about different monocytes and how CMML is defined. You need a certain level of white blood cells and monocytosis for a period of three months. In patients who suffer from this disease, monocyte levels are irregular. There is an increased fraction of classical monocytes. This means that monocyte phenotype can be used to detect the disease or an early ‘form’, called MDS. The speaker then went on to describe the genetic background of the disease. Several mutations occur in patients, 40 recurrently mutated genes were found in 49 patients. In CMML patients, the majority of the hematopoietic genes are mutated, there is a linear accumulation of mutations. The most mutated cells have a growth advantage and branching events can occur. CMML is a very severe disease and the main method of treatment is currently the use of hypomethylating agents. Baseline DNA methylation distinguishes responders and non-responders. Using this, you can predict CMML resistance to hypomethylating agents. After a conclusion, the presentation came to an end.

Actually, the majority of the presentation was new for me. I knew nothing about CMML before coming to this presentation. Moreover, this was my first HP Seminar, so I was excited to find out how these would take place. I learned a lot about the disease (how it is diagnosed, what is happening inside the body), but I also learned a lot about genetics and how several mutations can lead to a disease like this. Additionally, I found out a lot about different types of treatment and how the correct treatment for a patient is determined. The speaker had a very interesting flowchart describing how they determine what treatment is best suited. I really enjoyed the topic of the lecture, however it was often hard to follow. As someone with practically no background in the field, a lot of terms were knew to me and remained vague throughout the entirety of the seminar. The seminar was also given at quite a high speed. Despite this, I learned a lot. I really enjoyed finding out how different types of treatment are tested and implemented, given that I had very little knowledge about this.

seminar-1

This picture shows the survival rate of CMML patients, showing that CMML is truly a very severe disease.

In conclusion, this was a difficult seminar to follow but the topic was very interesting and educational. Even though medicine is maybe not the field I would really like to pursue, I would like to get to know more about this topic.

 

CRISPR Systems: Nature’s Toolbox for Genome Protection

Speaker: Prof. Dr. Jennifer Doudna

Department: UC Berkely

Subject: CRISPR Systems: Nature’s Toolbox for Genome Protection

Location: Wageningen University

Date: 30-9-2016

Author: Mirte Golverdingen

 

On Friday September 30th, I travelled to Wageningen to join the Lecture of prof. dr. Jennifer Doudna. She is one of the pioneers on the very promising CRISPR field. She started her lecture by explaining what CRISPR is and how it was discovered. CRISPR, Clustered regularly interspaced short palindromic repeats, are hallmarks of acquired immunity in bacteria.

The discovery of CRISPR started with curiosity on how bacteria defend themselves from viruses. 10 years ago, it emerged that many bacteria have an array of repetitive arrays in their genome, CRISPRs. In 2005 it was shown that the arrays contained virus RNA’s, this showed that CRISPR indeed could be a viral immune system. Over the next several years they did research to the way bacteria adapted to the viral DNA. It turned out that bacteria use a mechanism that adapts the viral DNA, then, crRNA is transcribed from this DNA and these CRISPR-RNAs can target complexes of virus DNA.

The Cas9 gene is the only gene that is necessary to fight of viruses by using CRISPR. From CRISPR to CRISPR systems there is much variation in how the DNA guides the DNA cutting work. These variations are classified into two classes, Class 1 and Class 2 systems.

Doudna did research to Class 2, consisting of CRISPR systems that contain one single enzyme responsible for detecting and destroying DNA. Cas 9 is a dual RNA guided DNA endonuclease. It is programmed by the crRNA, tracrRNA duplex and holds on to an unwound double stranded DNA helix. (See Figure 1) TracrRNA is important for creating crRNA but it also works in the complex with the crRNA. It trims away short molecules of RNA that are still functional. You can simplify this into a single guide instead of a dual crRNA-tracrRNA chimera. This Cas9 protein still worked.

honours-6

Figure 1: Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features. (A) (Top) In type II CRISPR/Cas systems, Cas9 is guided by a two-RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA (see fig. S1). (Bottom) A chimeric RNA generated by fusing the 3′ end of crRNA to the 5′ end of tracrRNA. Jinek & Chylinski et al. Science 337, 816 (2012).

Cas9 and its guide RNA act like a molecular scalpel to cut DNA. It searches on all the DNA in the cell to look if it can find DNA that match. When a match occurs the DNA is opened and cut with a double stranded break.

For the biotechnology, this is very interesting. Genome editing begins with dsDNA cleavage and results in non-homogenous end joining or Homologous recombination with a donor DNA between the two ends. Normally it is very hard to fix the double stranded break. However, the Cas9 is simple and cheap, it uses the DNA as donor so you can easily build in new genes. The CRISPR-Cas9 genome editing technology is useful because of the power of base pairing. It is a very convenient way to recognize DNA and to reprogram the specificity of the systems. All CRIPSR systems are based on this simple idea. Secondly, there are many applications of this technique in animal and plants. Moreover, it is a programmable DNA manipulation program with all the possibilities occurring from this idea.

The DNA target recognition is driven by pam motifs. Cas9 works by unwinding the DNA, however it does not hydrolize ATP. Cas9 first binds to PAM motives in the theta DNA curtains of the phage. PAM contact triggers the DNA unwinding, Cas9 is therefore a catalysator. In this way, the DNA can be unwound. The conformational change when Cas9 binds to the DNA is partly responsible for this DNA unwinding.

Cas9 searching mechanism is based on finding PAM motives, that are only seen in phage DNA. When there is recognition, unwinding finds place. The DNA will be cut and the virus is killed. Cas9 therefore does not slide along the DNA strand, instead it binds and searches very quickly and then leaves to another spot on the DNA.

I was very excited about this talk, it showed how the CRIPSR-Cas9 system worked. And it also showed something from research work done behind this revolutionary discovery. I hope there will be very renewing applications of this technique. The decade in this field will be even more exciting compared to the first decade.

New Frontiers in Nanofluidics Research

 

Speaker: Derek Stein

Department: Department of Physics, Brown University

Subject:

Location: TU Delft, Bionanoscience, Kavli Institute of Nanoscience

Date: 9-9-2016

Author: Mirte Golverdingen

Derek Stein gave his lecture to show what the new frontiers are in the Nanofluidics research.  He started with the research to use nanoscience for protein sequencing. The general idea was to combine mass spectrometry and nanotubes. The techniques are combined so they overcome the limitations of each technique. The nanotubes were already known by the public. Mass spectrometry, however, was a more obscure technique for us. Derek therefore gave some background to give more understanding of this technique.

Mass Spectrometry is a powerful tool; it can tell the weight of every single molecule. Therefore, it is an extremely well established technique. The tool separates ions by their mass to charge ratio (m/z) in vacuum. Nowadays it has such a high resolution that it can resolve ions differing in mass by a single Dalton.

Another new development is electrospray ionization. This is a soft ionization technique that can be used for proteins without destroying the conformation of the molecule. It works as follows; you take a little tube with a diameter of several dozens of nanometers. Through the tube you pass a fluid with the analyzing molecules. Then you put a strong voltage between the liquid and a metal part at the end of the evaporation chamber in the vacuum chamber. Small nanodroplets that leave the tip of the tube come into an evaporation chamber where there is a gas that dries them out. This results in forces that become stronger than the forces in the liquid droplets, so the droplets blow up and this results in smaller droplets. At the end of this process the droplets are shrinking into ions.

Stein used both techniques to take a mass spectrum of adenosine monophosphate. The resolution of this technique is very precise and it showed a peak at 346,4 FW. For Single-Ion Detection Stein used an electron multiplier, the Channeltron. In a process called secondary emission, a single ion can induce the emission of roughly 1 to 3 electrons. In the Channeltron a chain reaction of emissions is created, so one ion sprays out a lot of electrons. In this way, the signal of one ion is amplified. Therefore, Stein could detect the single ions that resulted from the electrospray ionization.

However, if you use mass spectrometry for sequencing proteins, peptide fragmentation is necessary. The polypeptide will break on the backbone, between the O and the NH atoms. This is ideal because the peptides will break into amino acids, however, you do not know how the protein breaks apart. More tests are therefore needed to discover the sequence of the protein, and, how longer the protein, how more tests are needed.

Stein’s lab wants to combine mass spectrometry with a source of single ions detection. If you can start your measurement by passing a single polymer across a nanotube, then you will break the polymer apart and use the mass spectrometer to measure which amino acid passes. The order of arrival will tell the exact sequence of the protein, without using further tests. Because this will take place in a vacuum chamber, no random motion will occur. In his lab, they have built a system to start test this idea. (See figure 1)

schematic-representation-of-the-setup

Figure 1: Schematic representation of the setup Stein has proposed. The protein gains access to the vacuum chamber by passing a nanotube. At the end of the tube fragmentation takes place. The amino acids then are recognized by mass spectrometry. The sequence of the amino acids is determined by the sequence of the protein that entered the nanotube. Liszweski, K. (2016). The Next Next Thing in Sequencing. GEN. 36.Retrieved from: http://www.genengnews.com/gen-articles/the-next-next-thing-in sequencing/5653?kwrd=Oxford%20Nanopore

Stein showed me a very exciting application in biology of nanotechnology. He also showed how important knowledge of biology, chemistry and physics is in the nanobiology field. I am very excited about new ways of thinking about protein sequencing and adapting nanotechnology in the biochemistry field. This is also something I want to learn more about. By adapting the protein fragmentation into the mass spectroscopy, you can determine the sequence of the protein more easily. This idea uses nanotechnology in the form of nanotubes and mass spectroscopy. The idea of Stein is very exciting and could be a revolution in the protein sequence world.

Nanobiology Honours Programme has started!

Dear reader,

Thanks for following our blog post!

This is the spot were we’d like to share the experiences of our Honours Programme students with you. Sharing the latest information on research within the Nanobiology field is essential for letting know the world about the importance of this exciting and new research field.

The Nanobiology Honours programma has started as of November 2014 for the first time ever. Six of our ambitious students with excellent study performances will have to attend several seminars from the Bionanoscience departments and Kavli Institute of TU Delft and the Biomedical Sciences departments of the Erasmus MC in Rotterdam.

Stay tuned for the latest exciting blogs of Kasper, Edgar, Jasper, Marloes, Max and Guus.

Best regards,

The Nanobiology Honours Programme team