Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1.
BACKGROUND
Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-beta (TGF-beta) 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.


RESULTS
We 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.


CONCLUSIONS
Our 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.
Garrido-Martin
Eva M
EM
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas and Centro de Investigación Biomédica en Red de Enfermedades Raras, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Blanco
Francisco J
FJ
Fernandez-L
Africa
A
Langa
Carmen
C
Vary
Calvin P
CP
Lee
Ursula E
UE
Friedman
Scott L
SL
Botella
Luisa M
LM
Bernabeu
Carmelo
C
D020121:5' Untranslated Regions; D030301:Activin Receptors, Type II / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D004285:Dogs; D006736:Horses; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D057079:Pongo; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
D020121:5' Untranslated Regions; D016329:Sp1 Transcription Factor; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II