Investigating the function of cis-regulatory elements for Runx1

Abstract

Detection of nucleotide sequence variations in patients of some diseases, provides strong evidence as to which genes and regulatory elements play a critical role in vivo. Acute Myeloid Leukaemia (AML) is a fast acting blood cancer acquired in the myeloid lineage. It can be caused by a number of genetic mutations or chromosome translocations, many of which are prognostically informative. However, because the molecular events that lead to the development and pathology of this leukaemia have thus far not been completely defined, difficulties arise in providing prognoses to AML patients without well-known mutations. Although AML subtypes are highly variable, the outlook is poor, with the majority of patients dying from the disease. The developmental transcription factor RUNX1/AML1 is a well-known leukaemia-associated gene. Runx1 is an important regulator of haematopoiesis in vertebrates; it is crucial for early myeloid differentiation, and plays a vital role in adult blood development. Disruptions to the RUNX1 gene are frequently associated with AML. Despite the well-established role of RUNX1 in haematopoiesis and disease contribution, the cis-regulatory mechanisms that modulate RUNX1 require further elucidation. In cases where no mutation is found in the coding sequence, there may instead be mutations in regulatory sequence, for example conserved non protein-coding DNA elements (CNEs) that affect gene function. Several research groups have identified an intronic Runx1 enhancer in mice, Runx1 +24 mouse conserved non-coding element (mCNE). The discovery of +24 mCNE as a haemogenic endothelial cell-specific enhancer in mouse and zebrafish embryos provided an insight into a mechanism for the tissue-specific transcriptional regulation of Runx1. Ng et al., (2010) then used comparative genomics to identify ten further putative Runx1 cis-regulatory elements. In light of the identified +24 enhancer, this project set out to investigate the functions other mCNEs detected by Ng et al., (2010). From the ten putative mCNEs described by Ng et al., (2010), five were selected for further investigation, with +24 acting as a positive control (-368, -101, +58.5, +110, +110.5-2 mCNEs). To investigate the putative cis-regulatory role of previously identified mCNEs in vivo, reporter constructs were used in zebrafish embryos, as well as human and mice cell lines. These reporter constructs provided evidence that the selected mCNEs do not act as ‘insulators’, which block transcription. However, potential enhancer activity was observed in the zebrafish assays from four of the mCNEs (-368, -101, +110, +24). Cell-specific enhancer assays indicated that one of the mCNEs, +110, might act as a heart specific enhancer, while another (-368 mCNE) could represent a blood specific enhancer. Cell culture reporter assays confirmed the enhancer activity of the +24 and +110 mCNEs. Further studies are required to validate functional roles of the other mCNEs and their interaction with Runx1. This study has provided novel evidence that +110 mCNE may act as a heart specific enhancer of Runx1, and showed that none of the mCNEs act as insulators. Preliminary data from zebrafish in this project suggests that further analysis of the mCNEs will provide insight into the elaborate orchestration of Runx1 regulation, as well as elucidate the genetic roles of RUNX1 and cis-regulatory elements in AML development