Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1
© Garrido-Martin et al; licensee BioMed Central Ltd. 2010
Received: 16 November 2009
Accepted: 29 June 2010
Published: 29 June 2010
Activin 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.
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.
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.
ALK1 (Activin receptor-Like Kinase 1) is a transmembrane type I receptor of the Transforming Growth Factor-β (TGF-β) superfamily of ligands, mainly found in endothelial cells. Its expression has been reported not only in highly vascularized tissues including lung, placenta, and heart [1, 2], but also at specific sites of epithelial-mesenchymal interactions , and in other cell types such as monocytes , microglia , skin fibroblasts , stellate hepatic cells , chondrocytes , neural crest stem cells  and more recently myoblasts . Nonetheless, most studies to date suggest that its major roles are related to the endothelial specific expression pattern. ALK1 is involved in angiogenesis [11, 12], and there is growing evidence indicating that it plays a key function in the arterial/venous differentiation during embryonic vascular development [13, 14]. It has been reported that ALK1 interacts with three ligands: with TGF-β1 and TGF-β3, in complex with the receptor type II (TβR-II) ; and with Bone Morphogenetic Protein 9 (BMP9), in complex with the Activin Receptor type IIA (ActRIIA) or the BMP receptor type II (BMPRII) . In the endothelium, circulating TGF-β signals from the lumen of the vascular vessel to the cytoplasm of the endothelial cell by interacting with its specific receptor complex. This complex consists of three different dimeric proteins: receptor type II (TβR-II), receptor type I (TβR-I) and an ancillary co-receptor (TβR-III: Betaglycan or Endoglin) . ALK5 is the predominant TβR-I in the majority of the cell types, but in endothelial cells ALK1 shares the TβR-I function with ALK5 in vitro. The significance of this apparent redundancy is explained because ALK1 and ALK5 signal in opposite directions, balancing the TGF-β signalling pathway in this cell type . ALK5 is able to arrest the cell growth, leading to a differentiated state in the maturation phase of angiogenesis, with formation of new extracellular matrix around the new vessel formed. ALK1 appears to play opposite physiological functions, since it is responsible for the events occurring during the activation phase of angiogenesis, including metalloprotease activation, proliferation of endothelial cells, and inhibition of differentiation . Thus, these complementary effects are mediated through different target genes of the two signalling pathways. ALK5 signals through Smad2/3 to regulate PAI-1 (Plasminogen Activator Inhibitor-1), Collagen I, or NOS-3/eNOS (Endothelial Nitric Oxide Synthase), whereas ALK1 signals through Smad1/5/8 to induce genes involved in proliferation such as Id1 (Inhibitor of differentiation 1), Id2 (Inhibitor of differentiation 2), Smad6, Smad7, ENG (Endoglin) or BMPRII . In addition, it has been shown that BMP9 is a quiescence factor for the microvasculature .
The gene encoding ALK1 (ACVRL1, Activin-A receptor type II-like kinase 1) spans 15,943 bp within the large arm of chromosome 12 (12q11-q14), coded on the positive strand (GeneID: 94). ACVRL1 cDNA was described first in 1993 simultaneously by two different groups showing a characteristic transcriptional start site (TSS), where the first exon is transcribed but not translated. To date, two different variants of mRNA transcripts have been described: variant 1 [GenBank:NM_000020.2]  and variant 2 [GenBank:NM_001077401.1] , both encoding the same ALK1 protein of 503 amino acids.
Mutations in ACVRL1 give rise to an autosomal vascular dysplasia called Hereditary Haemorrhagic Telangiectasia type 2 (HHT2) , while HHT type 1 is caused by mutations in ENG, the gene coding for the TGF-β co-receptor Endoglin . HHT1 and HHT2 are genetically dominant and mutant homozygosis is lethal, as confirmed in knock out mice for ENG and ACVRL1 [24–26]. Current estimates suggest that one in 5,000-8,000 people are affected by HHT . Patients are clinically diagnosed according to the Curaçao criteria , including nose bleeds, mucocutaneous telangiectases, internal arteriovenous malformations and familial inheritance. Around 90% of HHT patients have been genetically diagnosed as HHT1 or HHT2, and multiple mutations along exonic regions of ACVRL1 and ENG have been described . Because approximately 10% of HHT patients clinically diagnosed have unidentified mutations, the study of intronic sequences, splice sites and promoter regions of both genes is of critical importance. Moreover, haploinsufficiency is currently accepted as the basis for the pathogenicity of HHT  and therefore the understanding of the transcriptional mechanisms and the assessment of ways to increase the transcription rate may be crucial to identify strategies to counteract haploinsufficiency. While several reports have already described the organization and control of ENG promoter [30–33], very little has been done in relation to the ACVRL1 promoter. In this work, we have analyzed the 5'-proximal ACVRL1 promoter region, characterized new transcriptional initiation points, and analyzed the transcriptional mechanisms that regulate its expression.
Identification of novel TSSs for human ACVRL1 gene revealed by 5'RACE
Figure 1C shows the scheme of all the ACVRL1 transcript variants found in this work (mRNA1, 3 and 4) and in previous reports (mRNA1 and 2). The variant mRNA1 begins in the position + 1, followed by an exon of 278 bp (exon 2A) and then upon splicing of the longest intron (~ 5 kb) of ACVRL1, it continues with an exonic sequence containing the ATG start codon (exon 3). The mRNA2 has no exon 2A, and its exon 3 is longer in its 5'region, containing an additional 141 bp fragment. The starting ATG is located at the beginning of exon 3 (+ 5,058/+5,060), coinciding with mRNA1, therefore, both transcripts give rise to the same 503 amino acid protein.
The fragments obtained in the present work correspond to isoforms mRNA3 and mRNA4. The corresponding cDNA sequences have been deposited in GenBank database with accession numbers HM161905 and HM161906, respectively. Both of them reveal a cryptic exon placed upstream the + 1, resulting in two, instead of one, transcribed but not translated exons. This event gives rise to a rearrangement from 10 to 11 exons in the composition of the coding region of ACVRL1.
The human ACVRL1 promoter lacks TATA/CAAT boxes and has multiple Sp1 motifs
Main putative transcription factor binding sites found in the ACVRL1 promoter region
The ACVRL1 promoter region is highly conserved among different species
Basal activity of ACVRL1 promoter suggests the presence of possible positive and negative regulatory regions
Sp1 is critical for the ACVRL1 basal transcription
The next step was to dissect the Sp1 transcription induction in the different promoter constructs. S2 cells were co-transfected with Sp1 and with the different 5'-deleted constructs of the reporter pALK1 (Figure 6B). All constructs showed low basal transcriptional activities, which have been referred to value 1 and the stimulation is expressed as fold induction. When as little as 25 ng of the Sp1 expression vector was transfected, the activity levels of each construct were remarkably stimulated (between 26- to 158-fold induction values). It is worth mentioning that the maximum activity reached by the -422/+59 construct did not parallel its relatively low activity in endothelial cells, which have basal Sp1 levels (Figure 5B). This discrepancy is probably explained by the different background of transcription factors present in Drosophila embryonic versus human HMEC-1 cells.
To further assess the relevance of Sp1 in ACVRL1 transcription, Sp1 expression was abolished in mammalian Sp1-expressing cells. Thus, HEK293T cells transfected with siRNA targeted to Sp1 led to a marked decrease (more than 50%) of the ACVRL1 transcriptional activity in all the constructs (Figure 6C). These data confirm that Sp1 is essential for the transcriptional activity of the ACVRL1 promoter.
Chromatin immunoprecipitation of endogenous Sp1 bound to ACVRL1 promoter in HUVECs
Considering that the smallest fragment (-284/+59) maintains the majority of the promoter activity respect to the whole construct (Figure 5B), the nearest Sp1 sites upstream of the TSS (+ 1), appear to be critical for the ACVRL1 basal transcription. Interestingly, the -89/-56 fragment encompasses two adjacent Sp1 consensus elements, flanking a putative KLF6 binding site (Figure 7E). This fragment was selected as a probe for EMSA studies. A mobility shift appeared when nuclear extracts from HeLa cells (Sp1 rich) were incubated with the labelled probe and this binding was effectively competed with 100-fold excess of cold probe (Figure 7F). A supershift was observed in the presence of an antibody specific for Sp1 whereas anti-Sp3 or anti-NFκB antibodies did not affect the mobility shift, demonstrating the specificity for Sp1. The slight decrease of the retarded band in the presence of anti-Sp3 may be explained by the fact that Sp1 and Sp3 often bind to the same sites. Furthermore, single mutations of either Sp1 sites yielded retarded bands of a weaker intensity, suggesting that both sites are binding Sp1 independently. As negative controls, two Sp1 unrelated sequences, -823/-795 (containing consensus binding sites for other transcription factors, none of them homologous to Sp1 motifs) and AATT (double stranded poly dA-poly dT) were unable to compete with the probe, although with the AATT probe a slight non-specific reduction of the signal was observed.
The methylation status modulates ALK1 expression in endothelial cells
The characterization of the 5'-proximal ACVRL1 promoter fragment has allowed us to identify two new transcripts, upstream of the previously published TSS. Those transcripts include an upstream elusive exon, meaning that ACVRL1 may have several TSSs. Thus, ACVRL1, rather than showing a unique transcription start site, contains different regions with a variable probability of transcription initiation, a characteristic of TATA-less genes. Interestingly, this is a common feature shared between ACVRL1 and ENG. It is tempting to speculate that the existence of different points of transcription initiation may allow a more flexible regulation depending on the tissue, or the developmental stage of the cell. In certain tissues like placenta, two main types of transcripts have been reported [21, 22]. In HUVECs we have found two new transcripts that do not affect the open reading frame of the encoded protein. In the new transcripts, the first transcribed exon is not translated, and therefore no changes in the predicted protein sequence are observed. This observation emphasizes the importance of postranscriptional as well as transcriptional regulation mechanisms in the case of ACVRL1. The existence of different transcribed untranslated regions suggests regulation of ALK1 expression that involves the transcriptional rate, mRNA stability or interaction with other RNAs or RNA binding proteins. This feature is compatible with the high degree of conservation found among primates. Also, all the ACVRL1 transcripts described in human, orangutan, rhesus monkey and chimpanzee, seem to begin with an untranslated first exon. Thus, ACVRL1 may have important motifs for transcriptional regulation in the first exon. These motifs are included in the promoter fragment that has been subjected to study in this work.
Analysis of the basal promoter activity using luciferase-reporter experiments in a series of 5'-deleted mutant constructs revealed the existence of positive and negative regulatory elements. Interestingly, the shortest construct (-284/+59) displays a similar activity to that of the whole fragment (-1,035/+210). This finding suggests the existence of critical transcription factors binding to this area in order to enhance the transcriptional activity of the TATA-less ACVRL1 promoter. In agreement with this view, a high degree of conservation around the + 1 TSS among different species was found. The analysis also suggests the presence of a putative "repressor" segment between -422 and -284 positions, which inhibits ACVRL1 transcription. Interestingly, a similar region is found in the ENG promoter further upstream , something that deserves further investigation.
The in silico study of the ACVRL1 promoter sequence showed several important putative consensus elements, also found in ENG promoter [30, 32] that could be critical for the coordinated transcriptional regulation of these genes. This finding suggests that genes involved in the same functional pathway are controlled in a similar manner. Thus, ACVRL1 may be regulated by some of the transcription factors also controlling ENG expression. In this sense, because ENG is regulated by HIF-1, Smad, KLF6, Ets, or Sp1 [31–33, 37], it will be of interest to assess their functional implications in ACVRL1. Of note, three sequences matching HIF-1α motifs were found at positions -808/-792, -286/-274 and -236/-219 bp. The possibility that ACVRL1 could respond to HIF-1α is supported because hypoxia promotes angiogenesis, a process that is regulated by ALK1. In addition, multiple KLF consensus elements are dispersed along the ACVRL1 promoter region. Interestingly, this is another feature owing to the TGF-β receptor complex genes, in which the transcription is stimulated by KLF6 as an early injury response [18, 32]. On the other hand, the presence of multiple Ets sites, suggests regulation by MAPKs (Mitogen Activated Protein Kinases) . In this sense, in the functionally related gene, ENG, an Ets functional site was described in the proximal promoter . We also note that the presence of AP1 and NFκB sites suggests that ACVRL1 could be a target for the inflammatory response. In contrast to the ENG proximal promoter that contains abundant SBE motifs, only a single putative SBE was found at -270 bp position of the ACVRL1 promoter. Also, at variance with ENG, the ACVRL1 promoter does not contain any TGF-β-responsive element (SBE) in the neighbourhood of Sp1 sites. Moreover, the TGF-β receptor type I (ALK5) gene (TGFBRI), contains a SBE within the proximal promoter and both ENG and TGFBRI mRNA expression is upregulated by TGF-β [30, 39]. Thus, the proximal region of ENG is regulated by TGF-β through Smads, and a direct cooperation Smad/Sp1 giving rise to an important increase in transcription . Consistent with the lack of proximal responsive TGF-β elements within the ACVRL1 promoter, we found that its promoter activity was not regulated by TGF-β1 or BMP9. This does not exclude the possibility of a transcriptional regulation by TGF-β from SBEs placed further upstream, or by an indirect postranscriptional control through modifications of the receptor, which allow or prevent ALK1 to activate signalling through Smads. Addressing the functionality of all these transcription factors in the ACVRL1 promoter deserves an independent investigation. The present work has focused on the functional role of Sp1 on ACVRL1 basal transcription.
The most striking feature revealed by the in silico analysis of ACVRL1 is the presence of multiple Sp1 sites next to the different TSSs. Sp1 has been widely described as a general transcription factor involved in transcriptional basal mechanisms of gene promoters that lack TATA and CAAT core boxes. This is a shared characteristic among different housekeeping genes . Interestingly, this is also the case of several TGF-β receptors such as ENG and TGFBR1, whose transcription is driven by Sp1 [30, 31]. Further support for the involvement of Sp1 was obtained from the highly conserved alignment of the Sp1 consensus sites along the ACVRL1 promoter among several species that suggests a conserved biological relevance of Sp1 in the transcription of ACVRL1.
We have demonstrated here that Sp1 is a key factor necessary for the basal activation of ACVRL1 transcription. This effect has been observed both by Sp1 overexpression in cells lacking endogenous Sp1, and by interfering endogenous Sp1 with siRNA in mammalian cells. The large number of Sp1 consensus sites found in the ACVRL1 promoter, and the finding that small amounts of Sp1 can saturate its transcription, points out to a critical dependence on Sp1, and a fine tuning of ACVRL1 transcription by Sp1. Furthermore, a ChIP analysis in HUVECs after chromatin crosslinking revealed that Sp1 binds in vivo to the different consensus motifs along the whole proximal ACVRL1 promoter sequence studied (-1,032/+40). Thus, in endothelial primary cells growing in a rich medium, Sp1 may be binding to most of the specific motifs within the ACVRL1 promoter region. It can be speculated that the Sp1 binding to the starting transcriptional machinery complex is needed to ensure a transcription driven by Sp1 using different TSSs.
Because the closest region to the + 1 TSS is framed by the construction -284/+59 pALK1 and its transcriptional levels are similar to those of the -1,035/+210 pALK1 vector, probably the functional involvement of Sp1 for ACVRL1 transcription initiation is critical in this region. When searching the Sp1 consensus sites within this region, an interesting double site in the -89/-56 region was found. This Sp1 rich fragment contains two putative Sp1 consensus sites (-84/-78 and -67/-62) and, as shown in EMSA experiments, both sites are functional in binding to Sp1.
One key factor that modulates the Sp1 transcription dependency is the degree of methylation of the CpG islands contained within the Sp1 consensus elements. This is consistent with the observation that DNA methylation may interfere with the binding of Sp1 to DNA . In fact, we found that methylation of the -89/-56 Sp1 probe of ACVRL1 rendered this consensus motif inactive for Sp1 binding. It is well established that DNA methylation of CpG islands is an important mechanism for transcriptional regulation of multiple genes in mammals . However, most of these findings are related to proto-oncogenes, meaning that the hypermethylation is a protective mechanism for controlling the transcriptional switch to oncogenes. On the other hand, hypomethylation of tumor suppressor genes is a control mechanism for assuring its transcriptional rate. However, there are not many reports of genes non-related to cancer that could be controlled by their methylation state. For example, demethylation of certain promoters is involved in the return of somatic cells to previous undifferentiated stages of their cell lineage, and in the reprogramming to a new differentiating pathway in response to certain stimuli . In some genes, cytosine methylation of Sp1 sites has been reported as a marker of organ-specific expression and as a specific regulator of the expression . That could be the case of ACVRL1, in which demethylation of DNA in endothelial cells leads to a marked increase of ACVRL1 transcription, potentially because demethylation of CpG islands within the Sp1 sites is involved in ACVRL1 basal transcription.
ALK1 expression has been reported in several cell types, but its major roles are related to its predominant expression in endothelium. The ALK1 specific presence at the endothelial cell surface is tightly involved in the regulation of the TGF-β signalling pathway in balance with other type I TGF-β receptors . The endothelial specific expression may be explained by the presence within the proximal promoter region of ACVRL1 of consensus motifs for transcription factors (Ets, NFκB, Sp1 and KLFs) also shared by other endothelial specific genes such as ENG, PECAM1 ( Platelet Endothelial Cell Adhesion Molecule 1), VEGFR2 ( Vascular Endothelial Growth Factor Receptor 2), CDH5 (Cadherin 5), eNOS/NOS-3, or TIE2 (Tyrosine kinase with Immunoglobulin-like and EGF-like domains 2) [30, 31, 45–49]. In addition, the involvement of distal regulatory regions in the human ACVRL1, such as the one described in mouse that confers arterial endothelium-specificity, can not be excluded . Whether the degree of ACVRL1 methylation correlates with the endothelial specific expression of ALK1, remains to be explored.
Novel ACVRL1 transcripts have been identified, the ACVRL1 promoter has been characterized and its regulation by Sp1 has been demonstrated. Furthermore, a close dependence between ACVRL1 expression and the CG methylation degree was found. Future experiments to identify other trans-acting or trans-repressing factors in ACVRL1 regulation remain to be addressed.
5'Rapid Amplification of cDNA Ends (5'RACE)
Primers and probes used
5'RACE-adapter outer primer Fwd
ACVRL1 specific outer primer Rev
5'RACE-adapter inner primer Fwd
ACVRL1 specific inner primer Rev
pALK1 CLONING PRIMERS
Cloning primer (-898/-880) Fwd
Cloning primer (-587/-569) Fwd
Cloning primer (-422/-404) Fwd
Cloning primer (-284/-263) Fwd
Cloning primer (+42/+59) Rev
hALK1 primer Fwd
hALK1 primer Rev
h18S primer Fwd
h18S primer Rev
hSp1 primer Fwd
hSp1 primer Rev
hGAPDH primer Fwd
hGAPDH primer Rev
hId1 primer Fwd
hId1 primer Rev
pALK1 First region primer (-1032/-1012) Fwd
pALK1 First region primer (-844/-864) Rev
pALK1 Second region primer (-864/-844) Fwd
pALK1 Second region primer (-662/-682) Rev
pALK1 Third region primer (-510/-490) Fwd
pALK1 Third region primer (-260/-280) Rev
pALK1 Fourth region primer (-200/-180) Fwd
pALK1 Fourth region primer (+20/+40) Rev
pEPO primer Fwd (-66/-42)
pEPO primer Rev (+31/+9)
Sp1 sites WT pALK1 (-89/-56) Fwd
Sp1 sites WT pALK1 (-89/-56) Rev
Sp1 site MutA -84/-78 pALK1 Fwd
5'-CTGAGTTTT TT GGAGCGGCGCGGGGGTGGGTCCC-3'
Sp1 site MutA -84/-78 pALK1 Rev
5'-GGGACCCACCCCCGCGCCGCTCCA AAAAA CTCAG-3'
Sp1 site MutB -67/-62 pALK1 Fwd
Sp1 site MutB -67/-62 pALK1 Rev
pENG (-50 and -24 sites) Fwd
pENG (-50 and -24 sites) Rev
pALK1 (-823/-795) Fwd
pALK1 (-823/-795) Rev
AATT probe Fwd
AATT probe Rev
Real time PCR
For quantitative analysis, total RNA was isolated from cells using the RNeasy kit (Qiagen) and was reverse-transcribed using AMV reverse transcriptase (Roche Diagnostics). The resultant cDNA was used as a template for real time PCR performed with the primers shown in Table 2 using the iQ SyBR-Green Supermix (BioRad, Hercules, CA, USA). Amplicons were detected using an iQ5 real time detection system (BioRad). Transcript levels were normalized to 18S levels. Triplicates of each experiment were performed.
Cloning of the ACVRL1 promoter fragment
A genomic ACVRL1 fragment corresponding to the 5'-proximal region upstream the (+ 1) TSS was cloned into Sac I/Xho I sites of the reporter vector pGL2-luc containing the promoterless firefly luciferase gene (Promega, Madison, WI, USA). This construct comprises 1,244 bp and extends from the position 50,586,434 to 50,587,679 of the contig (GenBank: NC_000012.10. Reference Assembly). The construct was checked by sequencing with pGL primers 1 (Forward) and 2 (Reverse) (Promega) and the resulting sequence was identical to that of the GenBank.
Alignment among different species and in silico analysis of the ACVRL1 promoter
The human ACVRL1 promoter sequence was compared with the orthologous promoters in a set of animal species. The sequences to be compared were chosen by identifying the theoretical + 1 TSS and then aligning the human -1,035/+210 bp region. The accession numbers of the different contigs and the sequences used are: Mus musculus [GenBank:NT_039621] 62,243,937 - 62,245,182, plus strand; Rattus norvegicus [GenBank:NW_047784] 6,594,766 - 6,596,011, plus strand; Bos taurus [GenBank:NW_001495018] 123,378 - 122,133, minus strand; Canis familiaris [GenBank:NW_76284] 3,087,613-3,086,368, minus strand; Pan troglodytes [GenBank:NC_006479] 2,296,228 - 2,294,983, minus strand; Pongo pygmaeus [EMBL:413.52] 51,613,841 - 51,615,086, plus strand; and Macaca mulatta [GenBank:NW_001096621] 1,104,215 - 1,105,460, plus strand. For the in silico analysis of the putative response elements, we used the Genomatix MatInspector software tool: http://www.genomatix.de/products/MatInspector. The multiple sequence alignment was performed with the ClustalW2 software http://www.ebi.ac.uk/Tools/clustalw2, which generates a similarity score (values from 1 to 100) and provides a consensus proposal. Weblogo tool http://weblogo.berkeley.edu/logo.cgi was very useful to make the graphic representation of these consensus regions.
Generation of 5'deleted fragments of the human ACVRL1 promoter cloned into pGL2
Four different constructs with serial deletions of the ACVRL1 promoter were generated by PCR amplification, using primers designed to generate fragments with differences of ~150 bp between each other. All the sequences of the cloning primers are given in Table 2. Forward primers were at positions: -898/-880, -587/-569, -422/-404 and -281/-263. In all cases, the reverse primer was at positions: + 42/+59. The amplified fragments were: pALK1 -898/+59 (957 bp); pALK1 -587/+59 (646 bp); pALK1 -422/+59 (481 bp) and pALK1 -284/+59 (343 bp). The resulting products were purified and cloned into pCR2.1-TOPO-TA vector (Invitrogen, Carlsbad, CA, USA). Then, they were digested using Sac I and Xho I flanking restriction sites, inserted in pGL2-luc and checked by sequencing with commercial pGL primers 1 and 2 as described above.
Drosophila Schneider S2 cells were grown in Drosophila-enriched Schneider's (DES) insect medium (Sigma Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 0.1 μg/μl gentamicin and 2 mM L-glutamine. The human microvasculature endothelial cell line HMEC-1 was grown on 0.2% gelatin (Sigma Aldrich) pre-coated plates in MCDB-131 medium (Gibco, Paisley, UK) supplemented with 10% FBS, 2 mM L-glutamine, 1 ng/mL Epidermal Growth Factor (EGF) and 1 μg/ml hydrocortisone. Primary HUVEC were grown in EBM2 medium supplemented with EGM2 (Lonza, Walkersville, MD, USA) and containing 10% FBS. Human epithelial embryonic kidney HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. All the media were supplemented with both 100 U/ml penicillin-streptomycin.
DNA transfections and luciferase assays
Transfections of HEK293T, HMEC-1 and Schneider S2 cells were performed using the Superfect Reagent (Qiagen) following the commercial instructions. When required, transfected cells were treated for 3 hours with 1 ng/ml of TGF-β1 (R&D Systems, Minneapolis, MN, USA) or 16 hours with 0.5 ng/ml of BMP9 (R&D Systems) in the presence of 2% FBS. The expression vector pCIneo-Sp1 was used to transfect mammalian cells, whereas pPac-Sp1 was used to transfect Drosophila Schneider S2. The corresponding empty vectors were used as controls. Forty eight hours after transfection with the pGL2 reporter vectors, cells were harvested and the luciferase activities were determined in a TD-20/20 luminometer (Promega). In all cases, the pCMV-βGal vector was included in the transfections and its transcriptional activity was measured as an internal control. After normalization, the activity of the reporter constructs was referred to the basal activity as fold induction or as percentages with respect to controls.
Sp1 knock down
The human Sp1 small interfering ribonucleic acid (siRNA) was obtained from Santa Cruz Biotechnology (sc-29487, Santa Cruz, CA, USA). HEK293T cells were transfected with 5 pmoles of siRNA hSp1 or scrambled siRNA, using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Twenty four hours later, the pALK1 reporter construct and the Renilla normalization plasmid were transfected, using the Superfect Reagent (Qiagen). Luciferase activity was measured 24 hours after DNA transfection with the Dual Luciferase Assay System (Promega). Values were normalized to Renilla activity and values were referenced to the basal activity (100%) in each case. Lysates from these cells at 48 hours post-transfection were analyzed by semiquantitative RT-PCR using the primers indicated in Table 2 and by western blot using the rabbit polyclonal antibody anti-Sp1 (PEP2, SC-59, Santa Cruz Biotechnology) and monoclonal mouse antibody anti-β-actin antibody AC15 (A1978, Sigma Aldrich).
Chromatin immunoprecipitation (ChIP)
ChIP was performed with ChIP-IT Express kit (Active motif, Rixensart, Belgium), following the manufacturer's instructions. Briefly, HUVEC were grown to confluence and subsequently fixed with 1% formaldehyde. Cells were scraped in the presence of PMSF (phenylmethylsulphonyl fluoride) and lysed. Nuclei were separated using a dounce homogenizer and digested with enzymatic shearing cocktail for 15 min. One aliquot of this sheared chromatin was used as "input chromatin" and the rest was incubated with protein G magnetic beads and rabbit polyclonal antibody against human Sp1 PEP2 (SC-59, Santa Cruz Biotechnology) on a rolling shaker for 4 hours at 4°C. The positive control was incubated with rabbit polyclonal anti-human Histone 3 (ab8580, Abcam, Cambridge, MA, USA) and the negative control with serum anti-human IgG. Protein G magnetic beads bound to the immune complexes were pelleted, washed and bound proteins were eluted with the elution buffer provided with the kit. Then, the crosslinking was reversed and samples were incubated with proteinase K during 1 h at 37°C. Primers used for PCR were selected by mapping the whole promoter sequence, separated into four regions. The first region encompasses from -1,032 to -844 (188 bp); the second region from -864 to -662 (202 bp); the third region from -510 to -260 (250 bp) and the fourth region from -200 to + 40 (240 bp). Sequences of the four couples of primers are indicated in Table 2. For negative and positive control PCR, primers from ChIP-IT control kit human (Cat # 53010, Active motif) were used (data not shown). Measurement of the Sp1 binding was carried out using the following ratio of band intensities: (Sp1-IgG)/PInput.
Electrophoretic mobility shift assay (EMSA)
For the radiolabelled probe, the oligonucleotides were designed in the region -89/-56 of the ACVRL1 promoter, which includes two Sp1 consensus sites flanking a Krüppel-like factor-6 (KLF6) site. Competition experiments were carried out with five cold probes: i) -89/-56 wild type (WT); ii) -89/-56 with the site -84/-78 mutated; iii) -89/-56 with the site -67/-62 mutated; iv) -823/-795, containing consensus binding sites for other transcription factors, none of them homologous to Sp1 motifs; and v) AATT, an artificial competitor, double stranded poly dA-poly dT sequence, unrelated to the ACVRL1 promoter. The corresponding sequences are shown in Table 2. Probes were prepared by annealing complementary synthetic oligonucleotides followed by end labelling with [γ32P]dATP and T4 polynucleotide kinase. Nuclear extracts from HeLa cells were obtained from Promega (Cat # E3521). Approximately 5 ng (100,000 cpm) of the respective probe was incubated with 10 μg of nuclear extract and 2 μg/reaction of poly (dI-dC) for 30 min on ice. For competition experiments, a 100-fold excess of unlabeled double-stranded oligonucleotide was added. For supershift assays, protein extract and 1 μg of commercial antibody were preincubated for 60 min on ice prior to the addition of the remaining components of the binding reaction. Rabbit polyclonal antibodies anti-Sp1 (PEP2, SC-59), anti-Sp3 (SC-644) and anti-NFκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) (H-119, SC-7178) were purchased from Santa Cruz Biotechnology.
Positive control binding reactions were performed with a probe designed for the ENG promoter (-50/-24 Sp1 sites), which strongly binds Sp1 . Binding reactions were separated by nondenaturing 6% polyacrylamide gel electrophoresis in Tris-Borate-EDTA buffer at 4°C. Gels were dried, and visualized by autoradiography. EMSAs were repeated at least three times with similar results.
Treatment of cells with the demethylating agent 5'-aza-2'-deoxycytidine
CpG islands were detected using the software tool CpGplot http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html. HUVEC, HMEC-1, and HEK293T cells were treated with 5'-aza-2'-deoxycytidine (5-aza-dC; Sigma Aldrich) at a final concentrations of 1 μM or 5 μM for 7 days and the medium was changed every second day, according to Butta et al., . Finally, cells were lysed, total RNA was extracted with RNeasy kit (Qiagen) and the ALK1/Id1 ratio was measured by real time PCR. Primers used are shown in Table 2.
For functional experiments, after a one week of treatment with 5 μM 5-aza-dC, HMEC-1 cells were transfected with the specific reporter of the ALK1 pathway p(BRE)2-luc (BMP-responsive firefly luciferase reporter), which contains a small sequence with two copies of the regions (-1052/-1032)/(-1105/-1080) of the Id1 promoter .
In vitro methylation of promoters
Constructs -1,035/+210, -898/+59, -587/+59, -422/+59 and -284/+59 of ACVRL1 promoter and the reporter plasmids of Id1 promoter (pGL2-pId1) and the rat minimal prolactin promoter region -36 to + 37 surrounding a TATA box, (pXP2-TATAbox)  were methylated in vitro by the CpG methylase M.Sss I (New England Biolabs, Ipswich, MA, USA) in the absence or presence of the substrate S-adenosylmethionine. The methylation status was checked by digestion with the restriction enzyme Hpa II, which digests the target sequence CCGG but only when it is unmethylated (data not shown). Cells were transfected with 100 ng of the indicated mock-methylated or methylated ACVRL1 promoter reporter, 100 ng of pCIneo-Sp1 and with Renilla reporter vector. Luciferase activity was measured after 48 hours and normalized to Renilla activity. For electrophoretic mobility shift assays, the cold probe (-89/-56) of ACVRL1 promoter was subjected to in vitro methylation with the CpG methylase M.Sss I prior to competition with the radiolabelled probe.
Data were subjected to statistical analysis and results are shown as mean ±SD. Differences in mean values were analysed using Student's t-test. In the figures, the statistically significant values are marked with asterisks (*p < 0.05; **p < 0.01; ***p < 0.005; ns = not significant).
activin-A receptor type II-like kinase 1
Activin receptor-Like Kinase 1
Bone Morphogenetic Protein-9
Electrophoretic mobility shift assay
Endothelial Nitric Oxide Synthase
E26-Transformation-specific Transcription Factor
Fetal Bovine Serum
Hereditary Hemorrhagic Telangiectasia
Hypoxia Inducible Factor
Krüppel-Like Factor 6
Nuclear Factor of kappa light polypeptide gene enhancer in B-cells
5'Rapid Amplification of cDNA ends
Retinoid X Receptor
Smad Binding Element
Specificity Protein 1
Transforming Growth Factor-β
Transcriptional Start Site.
We thank Dr. Jonathan N. Berg (Clinical Genetics Ninewells Hospital and Medical School Dundee, UK) for providing the genomic DNA fragment of ACVRL1 promoter, and Drs. Tilman Sánchez-Elsner and Nora Butta for their helpful advices. This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (SAF2007-61827 to CB; SAF08-01218 to LMB, and predoctoral fellowship BES-2005-7974 to EMG-M), National Institutes of Health & National Center for Research Resources (P20-RR-15555 and HL083151 to CPV; DK56621 and DK37340 to SLF) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). The CIBER de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain.
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