Transcriptomic changes during differentiation of the leukaemia cell line THP-1 and the role of chromatin modifying enzymes

Abstract

During normal cell development, many genes are activated and repressed, usually through epigenetic mechanisms. These are modifications of the DNA and protein within the nucleus that result in changes in gene expression without alteration in DNA sequence. Key proteins for epigenetic modifications are the histone proteins bound to DNA in the nucleus. The best-characterised epigenetic complexes that modify histone proteins are the polycomb group proteins (PcG), comprising polycomb repressive complexes 1 (PRC1) and 2 (PRC2). The repressive modifications generated by these complexes can be removed, and the blocked genes reactivated, by enzymes that are the subject of this project. PRC1 repressive marks are removed by deubiquitinases USP12, USP16 and BAP1, whereas PRC2 marks are removed by demethylases KDM6A, KDM6B and potentially UTY. During the development of cancer, the regulation of many genes becomes abnormal, allowing the cells to escape normal growth restrictions. In this thesis, the expression of this set of chromatin-modifying enzymes in a leukaemia cell line was investigated. The FANTOM consortium has been helping to understand patterns of gene expression for over 10 years. The FANTOM4 dataset described changes in gene expression and promoter usage during differentiation of the THP-1 acute monocytic leukaemia cell line, using CAGE (Cap Analysis of Gene Expression) technology. This human monocyte-like cancer cell line can be stimulated with phorbol esters to halt proliferation and differentiate into macrophages. However, the FANTOM4 time course did not capture detailed mechanisms of regulatory factors in macrophage differentiation due to sparse time points and low read coverage.

The main aim of this project was therefore to repeat the time course with tighter time points and deeper sequencing of the transcriptome to develop a very precise picture of sequential activation of gene expression, transcription start site (TSS) usage and the activity of enhancers during the transition from proliferating monocytes to differentiated macrophage phenotype of the THP-1 leukaemia cell line, using CAGE.

The focus of this research was on the chromatin-modifying enzymes, but other key cell cycle and macrophage genes have also been examined. The differentiation time course was repeated in triplicate. RNA was extracted and CAGE libraries generated for 18 time points, including the 6 originally studied in FANTOM4. Sequencing results were analysed and normalised using bioinformatics tools. It was shown that analysing 8 samples on one Illumina HiSeq 2500 lane yielded enough read coverage to detect activity from even low expression TSSs, such as those associated with enhancer activity. Clusters of genes which were up- and downregulated at different time points during the differentiation process were identified and characterised. CAGE results for key genes encoding chromatin modifying enzymes and macrophage markers were validated by qRT-PCR. There was a rapid increase of histone demethylase KDM6B mRNA once differentiation was initiated. Histone deubiquitinase USP12 mRNA was also upregulated early in the process. Histone deubiquitinase BAP1 mRNA shows an interesting cyclic regulation pattern which was not seen in the more limited samples of FANTOM4.

These interesting chromatin-modifying enzymes and their close paralogues (deubiquitinases USP12, USP16 and BAP1, together with demethylases KDM6A, KDM6B and UTY) were investigated by bioinformatics and genetic tools. USP16 knockout THP-1 cell line was successfully created using CRISPR-Cas9 and its ability to differentiate into macrophages was examined using cell cycle analysis and CAGE sequencing. The USP16 knockout cell line, along with siRNA knock downs of USP12, USP16 and BAP1, was also compared to wildtype THP-1 differentiation using CAGE. Unfortunately, creating other mutant THP-1 cell lines was unsuccessful due to low THP-1 viability after single cell sorting. Investigating KDM6A, KDM6B and UTY using bioinformatics showed that UTY and KDM6A gene expression is positively correlated and this is disrupted in cancer samples. Gene expression and sequence comparison suggested that KDM6A and UTY are coregulated and may act in a similar way in histone demethylation.

In summary, the results in this thesis show the transcriptomic changes as the leukaemia cell line ceases proliferation and commences differentiation. Detailed examination suggests that histone modifications are important in the transition between proliferation and differentiation and provide better understanding of regulatory factors in macrophage differentiation and leukaemia.