Epigenetic editing:Towards sustained gene expression reprogramming in diseases

Veel ziekten zijn geassocieerd met verstoringen in genexpressie door epigenetische mutaties. In tegenstelling tot genetische mutaties, zijn epigenetische mutaties omkeerbaar. Hierdoor is het mogelijk gebleken om abnormale patronen van genexpressie om te draaien. Dit proefschrift heeft betrekking op de ontwikkeling van epigenetische editing-instrumenten om een verstoorde genexpressiepatronen in ziekten te herprogrammeren. Epigenetische editing bestaat uit een DNA-bindend domein (zink vinger, TALEs of CRISPR-Cas) gekoppeld aan het katalytische domein van epigenetische enzymen. Bovendien biedt gen-specifieke epigenetische bewerking de mogelijkheid om de regulatie van genexpessie te bestuderen. In dit proefschrift hebben we de mogelijkheid onderzocht om duurzame genexpressie modulatie te bereiken, door meerdere epigenetische markeringen te combineren en de rol van chromatin micro-omgeving in deze context te evalueren. Door gebruik te maken van verschillende DNA-bindingsplatforms gefuseerd met genexpressie activerende epigenetische enzymen, konden we in verschillende chromatine omgevingen, genexpressieherprogrammering behalen. Met behulp van deze techniek, waren we ook in staat om zowel de functie alsmede de mogelijkheid als nieuw therapeutisch doeleiwit van verschillende genen die betrokken zijn bij ziekten zoals kanker en COPD te onderzoeken. We gebruikten epigenetische editing om aan te tonen dat de overexpressie van TCTN2 een nieuw therapeutisch doeleiwit in kanker is. We hebben de eerste stappen gezet om de dubbele rol van RASSF1-isovormen bij de ontwikkeling van kanker te analyseren. Verder waren we in staat om aan te tonen dat remming van de activiteit van het slijmproductie-gen in COPD cellen inderdaad resulteert in minder slijmproductie. Onze aanpak heeft dus de belofte om verstoorde genexpressie patronen bij vele ziekte te kunnen herstellen.The reversible nature of epigenetic (mis)regulation, in contrast to genetic mutations, allows for the possibility of reverting abnormal patterns of gene expression at a molecular level. This thesis deals with the development of epigenetic editing tools in order to reprogram aberrantly expressed gene patterns in diseases. Epigenetic Editing consists of a DNA binding domain (Zinc finger, TALEs or CRISPR-Cas) linked to the catalytic domain of epigenetic enzymes. Furthermore, site-specific epigenetic editing provides the opportunity to study the regulation of gene expression. In this thesis, we addressed the possibility of achieving sustained gene expression modulation, by combining several epigenetic marks, and evaluating the role of chromatin microenvironment in this context. By using several DNA binding platforms fused to transcriptional activating epigenetic enzymes, we were able to show in different chromatin microenvironments, the possibility of achieving sustained gene expression reprogramming. Using this technique, we were also able to address the function and possible therapeutic use of several genes that are involved in diseases such as cancer and COPD. We used epigenetic editing to show that TCTN2 overexpression is a therapeutic target in cancer, and also to analyze the dual role of RASSF1 isoforms in the development of cancer. Furthermore, we were able to show that downregulation of a mucus production gene in cells might have therapeutic relevance for COPD. In conclusion, our approach has the potency to achieve gene expression reprogramming in diseases

Epigenetic Editing:On the Verge of Reprogramming Gene Expression at Will

Genome targeting has quickly developed as one of the most promising fields in science. By using programmable DNA-binding platforms and nucleases, scientists are now able to accurately edit the genome. These DNA-binding tools have recently also been applied to engineer the epigenome for gene expression modulation. Such epigenetic editing constructs have firmly demonstrated the causal role of epigenetics in instructing gene expression. Another focus of epigenome engineering is to understand the order of events of chromatin remodeling in gene expression regulation. Groundbreaking approaches in this field are beginning to yield novel insights into the function of individual chromatin marks in the context of maintaining cellular phenotype and regulating transient gene expression changes. This review focuses on recent advances in the field of epigenetic editing and highlights its promise for sustained gene expression reprogramming

Epigenetic editing:Towards sustained gene expression reprogramming in diseases

Veel ziekten zijn geassocieerd met verstoringen in genexpressie door epigenetische mutaties. In tegenstelling tot genetische mutaties, zijn epigenetische mutaties omkeerbaar. Hierdoor is het mogelijk gebleken om abnormale patronen van genexpressie om te draaien. Dit proefschrift heeft betrekking op de ontwikkeling van epigenetische editing-instrumenten om een verstoorde genexpressiepatronen in ziekten te herprogrammeren. Epigenetische editing bestaat uit een DNA-bindend domein (zink vinger, TALEs of CRISPR-Cas) gekoppeld aan het katalytische domein van epigenetische enzymen. Bovendien biedt gen-specifieke epigenetische bewerking de mogelijkheid om de regulatie van genexpessie te bestuderen. In dit proefschrift hebben we de mogelijkheid onderzocht om duurzame genexpressie modulatie te bereiken, door meerdere epigenetische markeringen te combineren en de rol van chromatin micro-omgeving in deze context te evalueren. Door gebruik te maken van verschillende DNA-bindingsplatforms gefuseerd met genexpressie activerende epigenetische enzymen, konden we in verschillende chromatine omgevingen, genexpressieherprogrammering behalen. Met behulp van deze techniek, waren we ook in staat om zowel de functie alsmede de mogelijkheid als nieuw therapeutisch doeleiwit van verschillende genen die betrokken zijn bij ziekten zoals kanker en COPD te onderzoeken. We gebruikten epigenetische editing om aan te tonen dat de overexpressie van TCTN2 een nieuw therapeutisch doeleiwit in kanker is. We hebben de eerste stappen gezet om de dubbele rol van RASSF1-isovormen bij de ontwikkeling van kanker te analyseren. Verder waren we in staat om aan te tonen dat remming van de activiteit van het slijmproductie-gen in COPD cellen inderdaad resulteert in minder slijmproductie. Onze aanpak heeft dus de belofte om verstoorde genexpressie patronen bij vele ziekte te kunnen herstellen

Stomatal and non-stomatal limitations on leaf carbon assimilation in beech (Fagus sylvatica L.) seedlings enduring moderate water stress under natural conditions

Seasonal variation and the differential effect of drought on photosynthetic parameters involved in carbon assimilation in forest species have been poorly studied under natural conditions. Limitations to diffusion and biochemical factors affecting leaf carbon uptake were analyzed in young beech seedlings (Fagus sylvatica L.) growing in natural gaps of a beech-wood at the southern limit of the species. Half of the seedling received periodic watering in addition to natural rainfall to reduce the severity of the summer drought which is typical in the area. Plant water status was evaluated by measuring predawn water potential. Basic biochemical parameters were inferred from photosynthesis-CO2 curves (A-Cc) under saturating light and chlorophyll fluorescence. The curves were established on three dates during the summer months Main variables studied included: stomatal and mesophyll conductance to CO2 (gs and gm respectively), maximum velocity of carboxylation (Vcmax) and maximum electron transport capacity (Jmax). The gm was estimated by two methodologies: the curve-fitting and J constant methods. Seedlings withstood a moderate water stress as the leaf predawn water potential (Ψpd) measured during the study was within the range –0.2 to –0.5 MPa. A mild drought caused gs and gm to decrease only slightly in response to Ψpd. However both diffusional parameters explained most of the limitations to CO2 uptake. Furthermore, non-stomatal limitations were also important limiting net photosynthesis as water stress increased. In addition, it should be highlighted that the biochemical limitations, prompted by Vcmax and Jmax, were related mainly to ontogenic factors, without any clear relationship with drought under the moderate water stress experienced by beech seedlings through the study. The results may help to further understand of the functional mechanisms influencing carbon fixation capacity of beech seedlings. This capacity may influence the total carbon balance of beech seedlings, and feed forward to their growth and survival under drought-prone environments such as those in the south of Europe.Peer reviewe

Stomatal and non-stomatal limitations on leaf carbon assimilation in beech (Fagus sylvatica L.) seedlings under natural conditions

Limitations to diffusion and biochemical factors affecting leaf carbon uptake were analyzed in young beech seedlings (Fagus sylvtica L.) growing in natural gaps of a beech-wood at the southern limit of the species. Half of the seedlings received periodic watering in addition to natural rainfall to reduce the severity of the summer drought. Plant water status was evaluated by measuring predawn water potential. Basic biochemical parameters were inferred from chlorophyll fluorescence and photosynthesis-CO2 curves (A-Cc) under saturating light. The curves were established on three dates during the summer months. The main variables studied included: stomatal and mesophyll conductance to CO2 (gs and gm respectively), maximum velocity of carboxylation (Vcmax) and maximum electron transport capacity (Jmax). The gm was estimated by two methodologies: the curve-fitting and J constant methods. Seedlings withstood moderate water stress, as the leaf predawn water potential (Ψpd) measured during the study was within the range –0.2 to –0.5 MPa. Mild drought caused gs and gm to decrease only slightly in response to Ψpd. However both diffusional parameters explained most of the limitations to CO2 uptake. In addition, it should be highlighted that biochemical limitations, prompted by Vcmax and Jmax, were related mainly to ontogenic factors, without any clear relationship with drought under the moderate water stress experienced by beech seedlings through the study. The results may help to further understanding of the functional mechanisms influencing the carbon fixation capacity of beech seedlings under natural conditions.Peer reviewe

TCTN2:a novel tumor marker with oncogenic properties

Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.</p

Project overview and update on WEAVE: the next generation wide-field spectroscopy facility for the William Herschel Telescope

We present an overview of and status report on the WEAVE next-generation spectroscopy facility for the William Herschel Telescope (WHT). WEAVE principally targets optical ground-based follow up of upcoming ground-based (LOFAR) and space-based (Gaia) surveys. WEAVE is a multi-object and multi-IFU facility utilizing a new 2-degree prime focus field of view at the WHT, with a buffered pick-and-place positioner system hosting 1000 multi-object (MOS) fibres, 20 integral field units, or a single large IFU for each observation. The fibres are fed to a single spectrograph, with a pair of 8k(spectral) x 6k (spatial) pixel cameras, located within the WHT GHRIL enclosure on the telescope Nasmyth platform, supporting observations at R~5000 over the full 370-1000nm wavelength range in a single exposure, or a high resolution mode with limited coverage in each arm at R~20000. The project is now in the final design and early procurement phase, with commissioning at the telescope expected in 2017.Comment: 11 pages, 11 Figures, Summary of a presentation to Astronomical Telescopes and Instrumentation 201