Phillips_whole_thesis.pdf (4.41 MB)
Regulation of ITGA6 and ITGB4 integrin genes by RUNX1 and epigenetic mechanisms
thesisposted on 2023-05-27, 11:06 authored by Jessica Phillips
Disruption to regulatory mechanisms controlling gene expression is a hallmark of leukaemia, with disruption to transcription factors being one of the most prevalent. By identifying the gene expression profile under the control of these transcription factors, and understanding how the target genes are regulated, critical insight can be gained into the role of these transcription factors in haematopoiesis, as well as their role in leukaemia development. Evidence presented here suggests that the RUNX1 transcription factor regulates the expression of the ˜í¬±6˜í‚â§4 integrin receptor in haematopoietic cells by controlling the integrin genes ITGA6 and ITGB4. Engagement of integrin receptors with extracellular matrix components of the bone marrow and haematopoietic tissues plays an essential role in haematopoiesis. Integrin expression is also altered in many leukaemias, however the regulation of integrin gene expression both in normal and disease states has remained largely unexplored. Data presented here identified ITGA6 and ITGB4 as novel target genes of the RUNX1 transcription factor in myeloid cells. RUNX1 was found to bind to the promoter regions of ITGA6 and ITGB4 in myeloid cells in ChIP assays. Furthermore, RUNX1 had a functional effect on both of the promoters in reporter assays. RUNX1 increased the activity of both promoters, while RUNX1-ETO, which is produced by a common chromosomal translocation in leukaemic cells, repressed promoter activity, consistent with its well-characterised role as a transcriptional repressor. While RUNX1 is commonly described as a sequence-specific DNA binding protein that binds to the consensus motif TGT/cGGT, it is becoming evident that the regulation of genes by RUNX1 is more complex than this and RUNX1 can regulate its target genes through a variety of mechanisms. Evidence presented here suggests that RUNX1 regulates the ITGA6 and ITGB4 integrin genes via two distinct mechanisms. RUNX1 was found to regulate the ITGA6 promoter through a consensus RUNX1 binding motif and RUNX1 activation of the promoter was dependent on this motif, in keeping with the traditional model of RUNX1 function described in the literature. In contrast, RUNX1 does not target the ITGB4 promoter through a consensus sequence motif and may be recruited indirectly to the promoter by other haematopoietic transcription factors. Furthermore, the data presented here suggest that efficient regulation of the ITGB4 gene may require interactions between the promoter and an upstream enhancer. RUNX1 was also found to interact with the ITGB4 enhancer, and similarly to the promoter, these interactions do not require a RUNX1 consensus binding motif and may involve recruitment by other transcription factors. Recent evidence suggests that the traditional model of RUNX1 function through a consensus binding motif may represent only a small proportion of RUNX1 target genes. Genome-wide analysis suggests that a significant proportion of RUNX1 recruitment to DNA occurs in the absence of consensus binding motifs, as shown here for ITGB4. To regulate gene expression, transcription factors must operate in the context of the nuclear chromatin environment. RUNX1 influence on gene expression is therefore also dependent on the chromatin environment at its target genes and its interactions with this environment. In the present study, epigenetic mechanisms were also found to contribute to the regulation of ITGA6 and ITGB4 gene expression in myeloid cell lines. ITGB4 expression was inversely correlated with DNA methylation of a large CpG island located at the promoter in KG-1a and Kasumi-1 myeloid cells. Furthermore, low levels of histone H3 and high levels of histone H3 acetylation at both ITGA6 and ITGB4 promoter regions was associated with higher expression of the genes in these cells. Expression of the ITGA6 and ITGB4 genes is likely to be a result of the interplay between transcription and epigenetic factors and in support of this, data presented here show that despite the presence of RUNX1 in KG-1a cells, ITGB4 is expressed at very low levels in these cells possibly due to high levels of DNA methylation at the promoter. This study has advanced our understanding of the mechanisms by which RUNX1 regulates its target genes and has identified distinct molecular mechanisms by which it operates. These findings may also be relevant to the mechanisms by which other transcription factors operate. Additionally, these findings suggest that RUNX1 disruption in leukaemia may have different effects on its target genes depending on how they are regulated normally by RUNX1. Additional studies are therefore required to further dissect the mechanisms by which RUNX1 regulates its target genes, and to further elucidate the repertoire of RUNX1 controlled genes. In addition, this study has provided insight into the regulation of integrin genes in myeloid cells, which is likely to have relevance to the regulation of these genes in other cell types and disease states.
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