Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1

Abstract

BackgroundActivin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1.ResultsWe have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1.ConclusionsOur results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity.

Trichloroethylene Induces Methylation of the Serca2 Promoter in H9c2 Cells and Embryonic Heart

“…These data suggest that specific methylation of the CpG dimer in position 66 may be important for regulation of Serca2a transcriptional activity. This result is supported by other studies reporting methylation of CpG dimers adjacent to SP1 DNA binding sites reduced SP1 binding [16,43,44]. In particular, Kitazawa et al [27] reported that in rat leukemia cell lines, methylation that was not within but adjacent to the two SP1 sites in the promoter significantly reduced cyclin D expression.…”

Activin receptor-like kinase 1 as a target for anti-angiogenesis therapy

“…The ALK1 gene contains 14 transcription factor binding sites for SP1, 1 Smad binding element, 7 for RXR dimers and 3 hypoxia inducible factor consensus binding sites. The promoter and enhancer regions are highly conserved throughout species and can be methylated to regulate gene expression [34]. The regulatory elements for arterial expression of ALK1 is located within a 9.3 kb genomic fragment, which contains the entire intron 2 and a 2.7 kb promoter region [35].…”

Genetic and Molecular Basis for Hereditary Hemorrhagic Telangiectasia

“…However, utility of this approach (as well as several other approaches discussed above) is predicated on the assumption that HHT vascular lesions result from haploinsufficiency and not dominant negative protein activity or a second somatic hit. Arterial endothelial-specific Acvrl1 expression in mice requires an enhancer in the equivalent of human ACVRL1 intron 1 [129], and transcription factors SP6 and KLF6 regulate ACVRL1 expression via sites in the proximal promoter [130,131]. In mice and zebrafish, Acvrl1 is regulated by blood flow/ mechanical force, although the underlying molecular mechanism is unknown [54,98].…”