Developing a protocol for in vitro assembly of chromatin using genome scale DNA molecules
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Epigenetics, the study of processes that change gene activity without altering the DNA sequence, has been observed to have a role in cancer, autoimmune diseases and neurodegenerative diseases. Epigenetic modifications can lead to alterations of the structure of the chromatin. To study how such changes affect the physical properties and dynamics of chromatin, in vitro assembled chromatin is a necessary tool as all the components are welldefined. In this Master’s thesis project, the aim was to develop a protocol for in vitro assembly of chromatin using DNA molecules in the kilo-basepair size range. The objectives were to optimize physical and chemical conditions in order to assemble chromatin of high quality, using human derived histone octamers together with lambda DNA and T7 DNA. The assembly was performed using a salt dialysis procedure followed by enzymatic assembly, using the enzymes nucleosome assembly protein 1 (NAP-1) and ATP-utilizing chromatin assembly and remodelling factor (ACF). The chromatin quality was assessed by either enzymatic digestion followed by gel electrophoresis or by using nanofluidic channels combined with fluorescence microscopy. The main goal was to assemble chromatin with periodically spaced nucleosomes. Using lambda DNA and an enzymatic digestion approach, it was possible to visualize DNA fragments on gels from agarose gel electrophoreses that corresponded to mononucleosomes. In one experiment, fragments corresponding to di- and trinucleosomes could also be visualized. The T7 DNA on the other hand yielded bands corresponding to mono- and dinlucleosomes to a higher degree, in comparison to lambda DNA, indicating higher chromatin quality. By using single-molecule nanofluidics in combination with fluorescence microscopy, it was possible to visualize co-localized DNA and histones. Overall, the chromatin assembled was not of the desired quality and it is therefore of interest to develop the protocol further.
chromatin assembly, lambda DNA, T7 DNA, nanofluidic channels, fluorescence microscopy