Changes in gene expression following DNA damage in a Non-Transformed Cell Line
thesisposted on 10.07.2017, 11:44 by Julian Wang
Post-transcriptional regulation of gene expression forms an essential part of the DNA damage response (DDR). It has been shown previously that following DNA damage initiated by a number of different agents, selective translational reprogramming occurs, with specific subsets of mRNAs displaying enhanced mRNA translation, despite an overall inhibition of protein synthesis. In the past, these studies were typically conducted using transformed cell lines, which have been shown to differ significantly in their DDR when compared to non-transformed cells. In this thesis, regulation of gene expression following DNA damage was investigated using the non-transformed MCF10A cell line through the use of the next-generation sequencing based technique, ribosome profiling. To this end, ionising radiation (IR) and ultraviolet radiation (UV-B) were tested as DNA damage sources, with UV-B proving to be more favourable. Using ribosome profiling, over 4000 significant differentially expressed genes were identified following UV-B irradiation, as well as a number of key pathways involved in this regulation. The use of ribosome profiling however, also presented technical issues. A significant proportion of contaminated reads (corresponding to 5’ tRNA halves) were found to be present in the sequencing data, which greatly reduced the effective read depth. Further investigations revealed that size exclusion columns used for the purification step of ribosome profiling selectively purified tRNA fragments (tRF). An alternative purification procedure using sucrose cushions resulted in reduced levels of tRF contamination. Another issue identified was sub-optimal RNase I digestion, a critical step in ribosome profiling. While a number of factors were investigated, the cause of the low level of RNase I digestion remained unresolved.