Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Speaker: Kirti Prakash

Department: Institute of BMolecular Biology, Mainz, Heidelberg University Heidelberg. Carnergie institution for science, Baltimore usa, current affiliation institute paris

Subject: Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Location: Bionanoscience Department, TU Delft

Date: 26-06-2017

Author: Mirte Golverdingen

A large spectrum of chromatin domains between DNA and the chromosome territory level remains unexplored.  We do understand the morphology of the DNA helix since the 1950’s and a decade ago Bolzer et al. showed the morphology of intra-chromatic departments of chromatins (see Figure 1).  However, the morphology and behavior of the region between single DNA morphology and intra-chromatic departments is still fairly unknown.  Genomic methods have already found a large spectrum of chromatin domains between the DNA and the chromosomal territory level, however their regulation is not known yet. To learn more of the regulation of gene expression, we have to obtain a better understanding of the unknown region.

 

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Figure 1: False color representation of all CTs visible in this mid-section after classification with the program goldFISH.

Adapted from: Bolzer, A., et al.  (2005). Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS biology, 3(5), e157.

Kirti Prakash studied these intermediate chromatin features by using single-molecule localization methods (SMLM). Classical microscopy methods, such as conventional light and electronic microscopy, are not able to identify intermediate chromatin features because of specificity and a poor resolution. Therefore, nanoscale imaging is required to describe chromatin complexity.

SMLM is the best method to study chromatin organization because it has the best resolution in a small area. From all SMLM methods, PALM/STROM has the best resolution and distance in xyz area. When asked if the time resolution had not to be taken into account, Prakash mentioned that time resolution is not important for the research he is doing. Moreover, he mentioned that the PALM/STORM microscope set-up technique is very simple, because no much alignment is needed.

However, the preparation of the buffers and fluorophores is very complicated, because the combination of buffer and fluorophores need to have a high photon expression number, prevents photo-bleaching and it has to blink for a long time. The search to a combination that has all three characteristics is time consuming and hard. Therefore, I think that PALM/STORM is not that better than other SMLM methods as Prakash suggested in his talk.

Dyes as Hoechst and DAPI undergo phot-conversion when binding to the minor DNA groove, therefore they are a good tool of chromatin studies for SMLM. The red shift of the fluorophore makes that you can exclusively excite the dyes that are bound to the DNA. This makes that the dyes do not bleach and that there are still a lot of signals. This high labeling density is required to study unknown structures.

According to Prakash, this method of direct DNA staining is better than previous methods based on antibody staining o histone proteins.  This is because you can have a 5 times higher labeling density. However, can we really compare the DNA-staining with histone labeling? Histones behave different from DNA itself, so are you not comparing apples with oranges?

By using the technique Prakash was able to study the hierarchical structure of the chromatin in a single experiment. He made 2D images of a 3D domain, so different in z-direction could not be measured. He showed that there were clusters of chromatin in the cell. However, is this really a fair conclusion if you have no idea of the clustering of chromatin in the z-direction.

By studying the clusters visualized, Prakash could research the chromatin complex morphology. He observed ring and rod like chromatin domains and displacement of active histone modification during stress. In mammalian cells, the chromatin complexes rearrange after stress. So, according to Prakash SMLM contributes to describe the spectrum of unknown chromatin domains between the nucleosome and chromosome level.

Prakash, however, did not convince me of the value of SMLM to the study of unknown chromatin domains. He compared the results of Histone labeling with his DNA staining assay, which was, I think, not fair. Moreover, his visualization of the chromatin complexes was in 2D without a verification that this 2D representation was valid. I think that a more validated study to chromatin complexes with this assay would have a better contribution to research on these complexes.

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.

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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.

3D organisation of the mammalian genome and its function

Speaker: Wouter de Laat
Department: Bionanoscience
Subject: 3D organisation of the mammalian genome and its function
Location: Delft University of Technology
Date: 2016-02-11
Author: Romano van Genderen

The seminar started by debunking a common myth, namely that 97% of all DNA, which is often considered “junk DNA” actually consists of all sorts of regulative elements and switches, which are turned off and on in specific tissues, causing the morphological changes. But not only the genetic information on this non-coding plays a regulative role, also the role it plays in changing the 3D organisation of the coding DNA plays a role.

The first experiment he presented that showed evidence for this was done by the Spitz lab. They showed that by moving a so-called transposon cassette, a piece of DNA that can be inserted in the genome, across the genome, the expression of specific proteins can be influenced. Sometimes a small step with the cassette leads to a large change in expression, while a large step does not make a huge difference. This was used to show that these two sites in the large step are separate on the genome, but topologically very close.

Another experimental method he presented was the 3C (chromosome conformation capture) method and other derived methods. 3C is based on formaldehyde linking the proteins on the DNA together into a sort of knot, then digesting them to cut the knot out. This is ligated and read to show which parts of the DNA are close together in space. He showed an example where this method was used to study the haemoglobin protein. Also he showed some derivative methods like 4C and Hi-C.

Afterwards he explained the basis of higher-scale genome folding in mammals. The main point he wanted to introduce were the so-called topologically active domains, also called TAD’s. These are loops in the DNA, separated from one another by a protein called CTCF. These domains also act as functional domains with promotors and enhancers relatively close by. A next step in folding is made by sorting the genome on the basis of activity. The active domains bind one another and so do the inactive domains. Afterwards, the inactive domains move towards the nuclear periphery. He showed a practical application of this by explaining how leukaemia originates.

Next he elaborated on the CTCF protein, which forms the anchor of a chromatin loop. The most important property of the protein is that it has a direction. In order for it to function properly and form a loop, the two proteins must not be in the same direction. This is better explained in the following image:

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Image 1: Image explaining the directionality of CTCF. Source: E. de Wit et al. CTCF binding polarity determines chromatin looping, Mol Cell, 60 (2015), pp. 676–684

Finally, he showed how 3C methods could be used for haplotyping, so to differentiate the maternal, paternal and foetal genome. This is because the more advanced 4C method can detect translocations in the genome. Because these C methods are based on location and not on sequence, a small difference in genome, like a few more SNP’s, are ignored. Using this haplotyping, a non-invasive form of prenatal diagnosis can be done using the fragments of foetal DNA found in the mother’s blood.

The thing I found most striking about this presentation is the situation that occurred during the questions. There was one person who was more of a proponent of the topological supercoiling model than the TAD model. Their discussions showed that sometimes one model is not sufficient to explain all the experiments.

Wnt/ß-catenin signaling controls cell reprogramming (and nucleosomes form clutches)

Bionanoscience Seminar, 08.05.2015

Speaker: Pia Cosma (Center for Genomic Regulation, Barcelona (Spain))

Author: Edgar Schönfeld

Pia Cosma’s research is focused on cell reprogramming. Her talk was divided into two major subjects that deal with cellular reprogramming: Wnt/ß-Catenin signaling and chromatin organization. Reprogramming can be achieved via nuclear transfer or direct reprogramming, which involves the introduction of the 4 pluripotency factors Oct4, Sox2, cMyc, and Klf4 into a somatic cell. A third option is reprogramming via cell fusion. Cell fusion itself is a natural process, which occurs for instance during the differentiation of muscle cells. However, cells can also be fused artificially in the lab, with the help of chemicals or electricity. If you fuse a stem cell with a somatic cell they give rise to pluripotent hybrid cell, because the pluripotent genome is dominant over the somatic one. (This process also happens in vivo and is involved in wound healing.) After such a fusion the pluripotency factor Oct4 is reactivated. In an experiment Pia tagged Oct4 with GFP to observe spontaneous cell fusions. She found out that reprogramming only occurs when the right amount of ß-Catenin is present, which is activated by the Wnt pathway.

In the last years she discovered a lot more details. She discovered that the ß-Catenin concentration fluctuates in embryonic stem cells (ESCs), which is absolutely important for the reprogramming of somatic cells via fusion with ESCs. Different levels of activity of ß-Catenin can regulate either pluripotency or differentiation. Furthermore she found out that repression of TSF3, a regulator of the Wnt pathway, can increase the reprogramming frequency several hundred times.

With that in mind, she wanted to know what happens to chromatin organization during reprogramming. As a result the textbook model of chromatin packing needs an important update! To be precise, the 3rd panel from the left in figure 1 is inaccurate. Pia discovered (using STORM) that the nucleosomes are not distributed uniformly over the genome. Instead nucleosome aggregate in small or big groups, which Pia calls ‘clutches’ (Fig.2).

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Figure 1: DNA Packaging, taken from http://www.shmoop.com/dna/dna-packaging.html

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Figure 2: New Model, source: http://www.cell.com/abstract/S0092-8674%2815%2900132-4

These clutches are separated by stretches of nucleosome-free DNA. Thereby the larger clutches form the silenced heterochromatin, because they contain more of the linker histone H1. RNA polymerases can still associate with the small clutches. In stem cells and during reprogramming the DNA is mostly covered with small clutches, which facilitates the docking of transcription factors and RNA polymerase. In somatic cells however, DNA is mostly packed around big silencing clutches. In an reprogramming event the cell has to switch from big dense to small loose clutches, but how is this achieved? Pia’s team hypothesized that this might occur through nucleosome sliding and removal, which is in accordance with their simulations. To summarize their findings the team created this video: