Transcriptional regulation of Elf-1: locus-wide analysis reveals four distinct promoters, a tissue-specific enhancer, control by PU.1 and the importance of Elf-1 downregulation for erythroid maturation

Ets transcription factors play important roles during the development and maintenance of the haematopoietic system. One such factor, Elf-1 (E74-like factor 1) controls the expression of multiple essential haematopoietic regulators including Scl/Tal1, Lmo2 and PU.1. However, to integrate Elf-1 into the wider regulatory hierarchies controlling haematopoietic development and differentiation, regulatory elements as well as upstream regulators of Elf-1 need to be identified. Here, we have used locus-wide comparative genomic analysis coupled with chromatin immunoprecipitation (ChIP-chip) assays which resulted in the identification of five distinct regulatory regions directing expression of Elf-1. Further, ChIP-chip assays followed by functional validation demonstrated that the key haematopoietic transcription factor PU.1 is a major upstream regulator of Elf-1. Finally, overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated that Elf-1 downregulation is necessary for terminal erythroid differentiation. Given the known activation of PU.1 by Elf-1 and our newly identified reciprocal activation of Elf-1 by PU.1, identification of an inhibitory role for Elf-1 has significant implications for our understanding of how PU.1 controls myeloid–erythroid differentiation. Our findings therefore not only represent the first report of Elf-1 regulation but also enhance our understanding of the wider regulatory networks that control haematopoiesis

A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity

The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)4 repeats. Deletion of one or two (GGA)4 motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter

EZH2 modulates angiogenesis in vitro and in a mouse model of limb ischemia

Epigenetic mechanisms may regulate the expression of pro-angiogenic genes, thus affecting reparative angiogenesis in ischemic limbs. The enhancer of zest homolog-2 (EZH2) induces thtrimethylation of lysine 27 on histone H3 (H3K27me3), which represses gene transcription. We explored (i) if EZH2 expression is regulated by hypoxia and ischemia; (ii) the impact of EZH2 on the expression of two pro-angiogenic genes: eNOS and BDNF; (iii) the functional effect of EZH2 inhibition on cultured endothelial cells (ECs); (iv) the therapeutic potential of EZH2 inhibition in a mouse model of limb ischemia (LI). EZH2 expression was increased in cultured ECs exposed to hypoxia (control: normoxia) and in ECs extracted from mouse ischemic limb muscles (control: absence of ischemia). EZH2 increased the H3K27me3 abundance onto regulatory regions of eNOS and BDNF promoters. In vitro RNA silencing or pharmacological inhibition by 3-deazaneplanocin (DZNep) of EZH2 increased eNOS and BDNF mRNA and protein levels and enhanced functional capacities (migration, angiogenesis) of ECs under either normoxia or hypoxia. In mice with experimentally induced LI, DZNep increased angiogenesis in ischaemic muscles, the circulating levels of pro-angiogenic hematopoietic cells and blood flow recovery. Targeting EZH2 for inhibition may open new therapeutic avenues for patients with limb ischemia

Molecular characterization of the proximal human endothelial nitric oxide synthase (eNOS) promoter

grantor: University of TorontoUnderstanding transcription initiation of the endothelial nitric oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present thesis focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of the constitutive expression of the human eNOS gene in vascular endothelium. Three tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domain I (PRD I) -104/-95 relative to transcription initiation), PRD II (-144/-115) and PRD III (-44/-25). The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1 and variants of Sp3, MAZ and YY1. Functional domain studies in 'Drosophila' Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1 and MAZ. The multiprotein complexes formed on the activator recognition sites within this region of the human eNOS promoter identify the existence of a constitutively active enhanceosome in vascular endothelium. Moreover, 'in vitro' DNase I footprinting analyses and nuclear run-off studies also indicate their important contributions to preinitiation complex formation. Surprisingly, episomal expression studies performed in endothelial and non-endothelial cell types revealed that eNOS promoter/reporter constructs lacked cell-specificity, suggesting that there exist cell-specific mechanisms that repress native eNOS gene expression in non-expressing cell types. We posited that DNA methylation played a role in this cell-specific expression. Sodium bisulfite genomic sequencing revealed a different methylation pattern of the eNOS promoter in endothelial versus non-endothelial cells. Transient transfection of 'in vitro' methylated promoter/reporter constructs revealed a possible role for methylation in transcriptional repression. DNA hypomethylation studies performed with 5-azacytidine, resulted in increased expression of eNOS mRNA in cell types that do not normally express the eNOS mRNA transcript. The role of epigenetic regulation in cell-specific eNOS gene expression is a novel finding in the vascular endothelium. We have identified novel concepts with respect to protein-DNA interactions, pre-initiation complex formation and chromatin-based epigenetic mechanisms in vascular endothelium.Ph.D

Estrogen-related receptor α1 up-regulates endothelial nitric oxide synthase expression

The human estrogen-related receptor α1 (ERRα1) is a member of an orphan receptor family closely related to the estrogen receptor. It has been demonstrated that estrogen modulates endothelial nitric oxide synthase (eNOS) expression through the estrogen receptor in endothelial cells. However, little is known about the relationship between ERRα1 and eNOS. In this study, we show that ERRα1 activates the estrogen response element (ERE) and eNOS promoter-dependent luciferase activity in COS-7 cells and bovine pulmonary artery endothelial cells. The endogenous ligand for ERRα1 has not been identified, but we show that these actions are dependent on serum constituents because ERRα1 fails to stimulate eNOS promoter-dependent luciferase activity in charcoal-treated serum. Furthermore, through the use of truncated eNOS promoter luciferase constructs, we demonstrate that the activation of eNOS transcription by ERRα1 is mediated via three regions: base pairs -1001 to -743, base pairs -743 to -265, and downstream from base pair -265 on the eNOS promoter. In addition, ERRα1 up-regulates eNOS mRNA and protein expression and stimulates eNOS activity in bovine pulmonary artery endothelial cells. These results suggest that ERRα1 has a potential role in the regulation of eNOS expression and may stimulate NO production by endothelial cells, which may in turn result in a protective effect against atherosclerosis