RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence

Abstract

Key Points• Generated the first comprehensive RUNX1b-specific transcriptome and binding profile in HE.• RUNX1b induces a cell adhesion and migration program prior to the downregulation of endothelial genes and the emergence of blood cells.During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion-and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development. (Blood. 2014;124(11):e11-e20)

The Role of Runx1 in Embryonic Blood Cell Formation

“…To overcome these challenges Lie-A-ling et al (Lie-A-Ling et al, 2014) used an alternative technique to chromatin immunoprecipitation called DNA adenine methyltransferase identification (DamID). DamID relies on the fusion of a transcription factor (such as Runx1) to the Escherichia coli DNA adenine methyltransferase (Dam).…”

“…DamID relies on the fusion of a transcription factor (such as Runx1) to the Escherichia coli DNA adenine methyltransferase (Dam). When the transcription factor binds DNA the fused Dam protein adds stable methylation tags to adenines within nearby GATC sequences allowing for identification of transcription factor binding sites without the need for antibodies (Lie-A-Ling et al, 2014). To identify Runx1 targets in HE, Lie-A-Ling et al established Runx1 −/− ES cell lines containing doxycycline inducible Runx1-Dam, and then differentiated the ES cells into HE.…”

“…To identify Runx1 targets in HE, Lie-A-Ling et al established Runx1 −/− ES cell lines containing doxycycline inducible Runx1-Dam, and then differentiated the ES cells into HE. Fortuitously, the inducible system was leaky, allowing for low levels of Runx1 expression in the absence of doxycycline that were not sufficient for EHT, but were sufficient for the detection of Runx1 occupancy by DamID (Lie-A-Ling et al, 2014). Comparison of the Runx1-DamID methylation and RNA-Seq datasets led to the identification of 235 genes that were both bound by Runx1 and differential expressed in HE cells generated from wild type and Runx1 −/− ES cells (Lie-A-Ling et al, 2014).…”

“…Fortuitously, the inducible system was leaky, allowing for low levels of Runx1 expression in the absence of doxycycline that were not sufficient for EHT, but were sufficient for the detection of Runx1 occupancy by DamID (Lie-A-Ling et al, 2014). Comparison of the Runx1-DamID methylation and RNA-Seq datasets led to the identification of 235 genes that were both bound by Runx1 and differential expressed in HE cells generated from wild type and Runx1 −/− ES cells (Lie-A-Ling et al, 2014). The expression of 80 of the genes was negatively correlated with Runx1 occupancy and 155 genes were positively correlated (Lie-A-Ling et al, 2014), consistent with Runx1’s ability to function as a transcriptional repressor or activator in the same cell type (Canon and Banerjee, 2003).…”

Runx transcription factors in the development and function of the definitive hematopoietic system

“…102 The Runx1 targets Gfi1 and Gfi1b are implicated in the downregulation of the endothelial program. 103 They do this through recruitment of the chromatin modifier LSD1 (a member of the CoREST repressive complex) to endothelial gene loci.…”