RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element
The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.
[Show abstract] [Hide abstract] ABSTRACT: The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear-structure building blocks in directly supporting nuclear function in a physiologically responsive manner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.0Comments 0Citations
- "This conformation, termed active chromatin hubs (ACHs), is mediated through the binding of transcription factors, and is only present when these genes are expressed. In hematopoietic stem cells (HSCs), the transcription factor RunxI is required for the interaction between the CD34 gene promoter and its downstream regulatory element [Levantini et al., 2011]. Transcription factor-mediated chromatin looping was demonstrated in erythroid cells. "
[Show abstract] [Hide abstract] ABSTRACT: Background Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown. Methodology/Principal Findings We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells. Conclusion/Significance The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.0Comments 12Citations
- "El Omari K reported that lmo2 functions as the scaffold for a DNA-binding transcription regulator complex, on condition knockdown of lmo2 leads to complete loss of PLM primitive hematopoiesis . Using transgenic mice carrying human CD34 PAC gene, Levantini identified a novel downstream regulatory element (DRE) that is bound by runx1 and is necessary for human CD34 in long-term (LT)-HSCs . Recently, Herbomel confirmed that zebrafish HSCs emerge directly from the aortic floor and this process is dependent on Runx1 expression . "
[Show abstract] [Hide abstract] ABSTRACT: To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.0Comments 31Citations
- "However, these data did not answer the question of whether CTCF binding or other mechanisms would mediate loop formation. Importantly, we recently described that targeted mutations of RUNX binding sites in a downstream regulatory element (DRE) of a human CD34 transgene caused the perturbation of the DRE-promoter interaction in transgenic mice (Levantini et al., 2011). Along with the functional models presented here, specific disruption of PU.1 binding in the URE of the endogenous PU.1 locus can be used to distinguish between correlation and causation of the transcription factor binding and chromosome looping necessary for gene activation. "
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