Epigenome Maintenance in Response to DNA Damage

Speaker:     Sophie Polo (University Paris Diderot)
Subject:      New insights into epigenome maintenance in response to DNA damage by real-time tracking of histone dynamics in human cells
Location:    Erasmus MC Rotterdam
Date:           Wednesday, 15.06.2015

Author: Edgar Schönfeld

The epigenome comprises a set of chemical modifications of the chromatin that regulate gene expression. It manifests itself in phenomena such as DNA methylations, histone modifications, and changes in chromatin folding. The epigenome can be passed to the next generation, although it changes dynamically over time. Sophie Polo’s work focusses on the preservation of epigenetic information that is encoded in histone modifications.
Histones enable the tight packaging of DNA in eukaryotic cells. A DNA strand is wound around octameric histone complexes and fixed via linker histones (This is called nucleosome). However, histones form a barrier to DNA repair. In order to access damaged DNA sequences, the appertaining histones first have to be removed or disassembled and subsequently be restored. This is generally referred to as ‘Access-Repair-Restore’ model. The question is whether the original histones or new histones are reincorporated after the repair has taken place. In the latter case, it is unclear if or how histone modifications are conserved.

Acces-Repair-Restore Model [1]

In order to maintain a stable epigenetic signature over time, epigenetic marks have to be faithfully restored after each DNA repair event. That includes the histone variant composition of a nucleosome, as well as its post-translational modifications. Already in 2006 Polo and colleagues observed that a certain histone variant, histone H3.1, is incorporated as newly synthesized substrate after nucleotide excision repair, following UV-induced damage. The deposition is mediated by the histone chaperone chromatin assembly factor 1 (CAF-1). This finding posed a challenge to epigenetic memory. There are two theories concerning the conservation of epigenetic information of removed histones: First of all, it could be the case that parental histone marks are transferred to the newly incorporated histones according to similar principles that apply to the inheritance of epigenetic marks in the process of replication. Secondly, it might be that no epigenetic information is transferred and the new histones serve as damage scars. These marks could be part of an adaption mechanism against further damage infliction. Polo went on to examine the link between DNA repair and histone removal and restoration. It was found that some other histone variants are replaced as well, while most histones just diffuse away during DNA repair and subsequently return to their original site. In particular, Polo said that 90% of the parental histones are conserved, which is good news for epigenetic integrity.
At this point of time, one can already say that Polo’s work has created important insights into epigenetic maintenance and I would not be surprised if her findings are incorporated in future textbooks.

[1] Polo SE, Roche D, Almouzni G. New histone incorporation marks sites of UV repair in human cells. Cell. 2006 Nov 3;127(3):481-93.


Synaptic origins of cerebellar disease

Speaker: Roy V. Sillitoe

Department: Jan and Dan Duncan Neuroloigcal Research Institute

Location: Erasmus MC Rotterdam

Date: 06-06-2016

Author: Carolien Bastiaanssen

In neurological movement disorders such as ataxia, dystonia, and tremor the patient suffers from involuntary muscle contractions. Roy Sillitoe and his colleagues aim to answer question such as how do circuits in the brain form and what are the electrophysical consequences when the wiring up does not go according to plan? In order to gain more understanding in how circuits form and function they use the cerebellum as a model system. In this way they also hope to get more insight into disease like dystonia and maybe even develop a treatment.


Figure 1. Location of the cerebellum.

The cerebellum is a part of the brain that is located at the bottom of the brain (Figure 1). This region of the brain is involved in movement, it is responsible for coordination, fine-tuning and motor learning. The cerebellum may also be involved in other cognitive functions like speech but this is still subject of debate. One of the dominant cell types in the cerebellum are the Purkinje cells (Figure 2). These neurons have parallel and climbing fibers through which they receive input. One of the main regions of the brain that provides input to the cerebellum is the so called inferior olive. All these input signals are integrated and the output is send to the deep cerebellar nuclei. Purkinje cells have two types of action potentials: single and complex. In this way the Purkinje cells provide feedback for motor coordination.

purkinje cell

Figure 2. Purkinje cell in the cerebellum.

Researches often make mouse models of diseases. However, most of the mouse models that were based on human genetic studies did not display dystonic symptoms. Now Sillitoe and his colleagues work on a different mouse model and this model does show dystonic symptoms. It is known that cerebellar nuclei function is abnormal in dystonia and that dysfunction of Purkinje cells is linked to this disease. The type of abnormalities implicate that the inferior olive works as a trigger for dystonia. So Sillitoe and his colleagues asked themselves whether the inputs to the motor system could be at fault in dystonia. The inferior olive to cerebellum connection is a good candidate because it’s thought to control timing and/or error correction during movement and learning. This gave rise to the idea of using the inferior olive to cerebellum circuitry as an inroad for designing a more flexible model for understanding dystonia. The goal (or rather wish list) was to generate a genetic mouse model that exhibits dystonia and that is experimentally tractable for development, behavior, anatomy and physiology.

They started with the hypothesis that selectively silencing developing olivo-cerebellar synapses will cause circuit malformations that induce dystonia in mice. They could not just cut the entire olive because that would have severe consequences for the development of the mice. It is however difficult to target the inferior olive because its gene expression pattern overlaps with connected regions. They therefore chose to use a unique intersection of gene expression in the inferior olive (Ptf1a and Vglut2). This caused the inferior olivary neurons to release empty vesicles and thus no response is triggered. The mice indeed showed symptoms of dystonia and the mice were further studied by amongst others electromyography (EMG), measuring local field potential (LFP) and single-unit recordings.

The next step was to test how the mice could be treated. Sillitoe and his colleagues for example used deep brain stimulation (DBS) which is already used in the clinic to treat patients with Parkinson’s disease. It turned out that during DBS the mice were free of any symptoms but when after DBS the symptoms returned.

All in all it was an interesting talk by Roy Sillitoe but it was quite difficult to follow due to the speed with which he went through everything and because of the many medical terms that I am not familiar with. In the end however the basics were clear to me and the model they developed seems very promising.


Synaptic origins of cerebellar disease

Speaker: Roy V. Sillitoe
Department: Department of neuroscience
Subject: Synaptic origins of cerebellar disease
Location: Queridozaal (Erasmus MC)
Date: 06-06-2016

Author: Kristian Blom

At the sixth of July I visited a seminar given by Roy Sillitoe, assistant professor at the Baylor College of medicine in Houston. Roy Sillitoe’s major goal of research is to determine the biological bases underlying complex pediatric neurological diseases. He did his PhD at the university of Calgary. After that he did a short Postdoc at Oxford. During this seminar, dr. Sillitoe discussed his recent unpublished work about the synaptic origins of cerebellar diseases.

Dr. Sillitoe and his group use the cerebellum as a model system for understanding how circuits form and function. Molecular damage to the cerebellum cause different motoric problems: Tremor, ataxia and dystonia are examples of such diseases. Now the question is: How do these diseases arise from a single circuit? How does one single cell type give rise to such diverse set of diseases? It turns out that this is not quite simple from a molecular point of view.

Figure 1 – Cytoarchitecture and connectivity in the cerebellum. (A) Mouse brain shown from a lateral view with the cerebellum highlighted in color. (B) The basic cerebellar circuit is comprised of granule cells, Purkinje cells, stellate and basket cell interneurons, and deep nuclei.                                                                                                                                                                                                     (Source: Roy V Sillitoe et al. (2013) New roles for the cerebellum in health and disease. Frontiers in Systems Neuroscience 7:83.)

The disease Dr. Sillitoe focuses on is dystonia, a neurological movement disorder in which sustained muscle contractions cause twisting and repetitive movements or abnormal postures. Dystonia is the third most common cerebellum disease, behind Parkinson and tremor. Dystonia can either be inherited or acquired. In case of the former, dystonia can be broadly divided in two major categories: DYT1 early onset generalized dystonia and non-DYT1 early onset dystonia. DYT1 generalized dystonia is known to be caused by a specific mutation in the DYT1 gene.

Doing research to dystonia requires mice which you can genetically modify to express the dystonia disease. Unfortunately, when you mutate those genes in mice which are normally mutated in people with dystonia, they won’t express the dystonia phenotype at all. But if mutations of these genes doesn’t work, how do we get the disease phenotype in mice? Well, recent evidence suggests that the cerebellum is not only involved in dystonia, but it can instigate dystonia. In other words: it may not be mutations in the cerebellum itself that cause dystonia, but the signals which are transferred through the cerebellum that are the ‘inputs’ to the motor system might be at fault in this disease. So we don’t have to do anything with mutations of the cerebellum, but with the signals passing through the cerebellum. Luckily, the inferior olive, the largest nucleus situated in the lower portion of the brain stem, has a powerful and unique excitatory connection to the cerebellum. But can we use the inferior olive of the cerebellum circuitry as an inroad for designing a more flexible model for understanding dystonia? Dr. Sillitoe tackled this challenge by selectively blocking neuro-transmission to the cerebellum. His hypothesis about this experiment was: Selectively silencing developing olivo-cerebellar synapses will cause circuit malformations that induce dystonia in mice. And it turns out he was right!

After Dr. Sillitoe succeeded in showing that selectively blocking of signals to the cerebellum resulted in a dystonia phenotype, a new question came around the corner: Is a bad cerebellum signal worse than no signal at all? This question was answered by inactivating the cerebellum in mice using an osmotic pump filled with lidocaine, which is a chemical agent that can inactivate the cerebellum. An osmotic pump provide researchers with a method for controlled and continuous agent delivery in vivo. Mice which first displayed dystonia, totally lacked the dystonia phenotype after the osmotic pump was activated. Hence, this suggest that a bad cerebellum signal is indeed more worse than no signal at all.

Finally, I want to end with something I found rather cool: Deep brain stimulation (DBS). DBS is an effective therapy for neurological and neuropsychiatric diseases that uses electrodes which are inserted in the brain. Dr. Sillitoe and his group tried to recover movement by DBS of the cerebellar. And guess what… they succeeded! It turns out that DBS directed to the cerebellum significantly improves mobility. But how DBS does this is so far unknown. Perhaps that in a couple of years some nanobiologists, with their knowledge about systems and signals (which is very relevant for DBS), have the answer to this question.