Human DNA consists of three billion base pairs, which corresponds to a total length of approximately two metres. DNA must be compressed 200,000-fold in order to fit into the tiny nuclei of mammalian cells. The thread-like complex of DNA and proteins is called chromatin. Although chromatin has been widely studied, relatively little is yet known about the spatial and temporal organisation of chromatin in interphase cells.
Christof Gebhardt, a biophysicist from Ulm University, is using a new microscopy method to study the principles that underlie the organisation of chromatin. The European Research Council (ERC) will be providing funds totalling 1.5 million euros for a period of five years, which the 36-year-old will use to establish a research group on ”Single Molecule Mechanisms of Spatio-Temporal Chromatin Architecture” (ChromArch).
Gebhardt, who has been professor for experimental physics at the Institute of Biophysics at the University of Ulm since 2013, wants to obtain new insights into chromatin architecture and gene regulation, and potentially also into the development of hereditary diseases. He is using RLSM, a technique that he developed as a post-doc in the laboratory of Prof. X. Sunney Xie at Harvard University (2010-2013).
RLSM stands for reflected light-sheet microscopy and is based on light-sheet fluorescence microscopy, which was further developed by the Frankfurt physicist Ernst Stelzer and chosen by the scientific journal Nature as Method of the Year 2014. RLSM can image single molecules deep in the cell by specifically illuminating a thin slice of the cell. The camera, which records the light emitted by the fluorophores, is set at a perpendicular angle to the light sheet so that only the plane of interest is illuminated.
Xie was the first ever researcher to show that single fluorescent proteins can be studied in bacteria. After his PhD at the Technical University in Munich, Gebhardt joined Professor Xie at Harvard University where he wanted to work on transferring the technique from bacterial to human cells, which are many times bigger than bacterial cells. Using light-sheet microscopy, Gebhardt managed to selectively illuminate thin (up to 500 nm) slices, thereby overcoming the problem of autofluorescence, which tends to mask the signal that a researcher is seeking. As light-sheet microscopy was developed for organisms such as zebrafish, which have embryos that are about 50 times bigger than human cells, Gebhardt had to find a way to observe single biomolecules in much smaller human cells. The solution he found was to choose a coaxial arrangement of objectives as well as equipping the vertical objective with a small mirror to avoid the sterical limitations that normally occur with vertical objectives.
Gebhardt was able to establish the RLSM technique at Harvard. Previously, approaches such as confocal microscopy, spinning disc microscopy and two-photon excitation for application to single molecules failed. Although the resolution achieved by these methods was as high as that of RLSM, they were unsuitable for studying living cells. At Harvard, Gebhardt was specifically interested in the dynamic interaction between transcription factors and DNA. The docking of transcription factors to specific DNA signalling elements “switches” genes on. Using RLSM, Gebhardt found that transcription factors reside on DNA for no more than a few seconds while the actual transcription process in mammalian cells takes around 10 to 30 minutes. Gebhardt’s project at Harvard dealt specifically with ways to visualise the movements of transcription factors.
The ERC project involves the development of an RLSM-based method that will enable the researchers to study the structure of chromatin during interphase cells, of which little is yet known. Cell nuclei are fairly small and DNA needs to be packaged into a smaller volume to fit into them. To achieve this condensed form, the DNA winds itself around proteins (histones), thereby forming nucleosomes. Spiral structures, called soleonids, result from the helical winding of several nucleosome strands.Chromatin is far from being a rigid structure. Instead, it is quite a dynamic system, which, according to current knowledge, is also involved in controlling gene activity. During interphase, a phase of the cell cycle which takes several hours, the chromosomes occupy so-called chromosome territories with specific structural properties.
Special sequencing methods (e.g., chromatin conformation capture) have provided an initial idea about the structural properties and spatial organisation of chromatin in a cell’s natural state. These methods chemically freeze the cell and enable the identification of DNA regions in proximity to one another. The chromatin conformation capture technique requires a large number of cells; the calculation of a mean value gives the researchers an approximate idea of which DNA regions interact more frequently than others. One such example is enhancer and promoter sequences that control the transcription of DNA into RNA. Enhancer sequences are usually located far from the genes. When the DNA folds, these sequences are moved spatially closer to other regulatory regions.DNA can only be transcribed correctly when the enhancer sequences are spatially so close to the promoter that they are able to interact. Gebhardt therefore assumes that chromatin territories need finely balanced architectures in order to enable several thousand genes to be transcribed. Some DNA regions need to interact or come into close proximity with one another, others have to remain apart. Bulk experiments usually only allow general probability statements on chromatin structure to be made, which is why Gebhardt is keen to apply single-cell approaches.
Gebhardt wants to find out what movable chromatin within a certain territory is like. Electron microscope images of chromatin show that it is organised into distinct domains: condensed heterochromatin and lightly packed euchromatin. Gebhardt believes that more distant DNA regions are found in the euchromatic regions. He will use the ERC funds to find out how the heterochromatic and euchromatic regions move in relation to one another.Gebhardt’s goal is to find out why cells arrange their DNA in the way they do, how the loops are formed and what their function is. Using RLS microscopy, Gebhardt wants to visualise DNA by labelling it with fluorescent dyes in order to elucidate its structure. He will initially use human cell lines (most likely from different tissue types) because they are easily handled in the laboratory.