Epigenetic Regulation of Intestinal Stem Cells by Heterochromatin Mark H3K9me3

The intestinal stem cells (ISCs) adapt in response to environmental factors and continually proliferate to renew the mammalian intestinal epithelium due to its rapid turnover. Overall, intestinal homeostasis is maintained by the differentiation and self-renewal of ISCs, which are regulated by different mechanisms, including epigenetic histone modifications. Earlier studies in the host laboratory have shown that the histone methyltransferase Su(var)3-9 is essential in the nutrient-induced activation of intestinal stem cells. Su(var)3-9 specifically trimethylates histone H3 on lysine 9 (H3K9me3), which is a repressive histone mark, responsible for transcriptional silencing at heterochromatin regions. It influences stem cell maturation, lineage specification, and many other cellular processes. However, the precise mechanisms behind its function in ISCs remain unknown – that knowledge is important for understanding the development of many diseases, including cancer and metabolic disorders. This thesis aimed to investigate the distribution of the heterochromatin mark H3K9me3 in the intestine with an emphasis on ISCs, using the Drosophila midgut and mouse intestinal organoids as models. Confocal microscopy was used together with cell-type-specific fluorescent staining, to obtain the expression of the H3K9me3 specific histone methyltransferase Su(var)3-9, in the midgut. An antibody was used for the detection of H3K9me3 distribution along the anterior/posterior axis in Su(var)3-9 overexpressed flies. Additionally, DNA adenine methyltransferase identification (DamID) was applied in order to find target genes of the H3K9me3 regulation in the genome with the specific chromo domain of M-phase phosphoprotein 8 (MPHOSPH8) that binds to H3K9me3. The number of lineage-labeled differentiated enterocytes was shown to be locally higher in the Su(var)3-9 overexpressed flies compared with the control, although the flies were on starvation without nutrient-induced activation. Moreover, the number of lineage-labeled progenitor cells was not remarkably altered between the samples. However, the intensity of H3K9me3 was significantly higher throughout the whole midguts in the Su(var)3-9 overexpressed flies in comparison to the control. According to one replicate, the DamID in mouse intestinal organoids revealed that the peaks of H3K9me3 were divergent between the samples grown in different conditions. The first sample was assumed to contain more ISCs, whereas the other one was assumed to contain more differentiated intestinal cells. According to my results, the Su(var)3-9 overexpression drives the stem cells against the differentiation of enterocytes. Furthermore, the MPHOSPH8 chromo domain in the organoids was successfully applied in DamID; thus, more replicates should be prepared for additional analysis, because I found several potential target genes of H3K9me3. In the future, it is important to further study the epigenetic regulation of ISCs, for applying the epigenetic marks as targets for the treatment of many human pathophysiological conditions, such as cancer, obesity, and metabolic disorders.