BACKGROUND
alk <dig>  is a transmembrane type i receptor of the transforming growth factor-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  <cit> , but also at specific sites of epithelial-mesenchymal interactions  <cit> , and in other cell types such as monocytes  <cit> , microglia  <cit> , skin fibroblasts  <cit> , stellate hepatic cells  <cit> , chondrocytes  <cit> , neural crest stem cells  <cit>  and more recently myoblasts  <cit> .
nonetheless, most studies to date suggest that its major roles are related to the endothelial specific expression pattern.
alk <dig> is involved in angiogenesis  <cit> , and there is growing evidence indicating that it plays a key function in the arterial/venous differentiation during embryonic vascular development  <cit> .
it has been reported that alk <dig> interacts with three ligands: with tgf-β <dig> and tgf-β <dig>  in complex with the receptor type ii   <cit> ; and with bone morphogenetic protein  <dig> , in complex with the activin receptor type iia  or the bmp receptor type ii   <cit> .
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 , receptor type i  and an ancillary co-receptor   <cit> .
alk <dig> is the predominant tβr-i in the majority of the cell types, but in endothelial cells alk <dig> shares the tβr-i function with alk <dig> in vitro.
the significance of this apparent redundancy is explained because alk <dig> and alk <dig> signal in opposite directions, balancing the tgf-β signalling pathway in this cell type  <cit> .
alk <dig> 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.
alk <dig> 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  <cit> .
thus, these complementary effects are mediated through different target genes of the two signalling pathways.
alk <dig> signals through smad2/ <dig> to regulate pai- <dig> , collagen i, or nos-3/enos , whereas alk <dig> signals through smad1/5/ <dig> to induce genes involved in proliferation such as id <dig> , id <dig> , smad <dig>  smad <dig>  eng  or bmprii  <cit> .
in addition, it has been shown that bmp <dig> is a quiescence factor for the microvasculature  <cit> .
the gene encoding alk <dig>  spans  <dig>  bp within the large arm of chromosome  <dig> , coded on the positive strand .
acvrl <dig> cdna was described first in  <dig> simultaneously by two different groups showing a characteristic transcriptional start site , where the first exon is transcribed but not translated.
to date, two different variants of mrna transcripts have been described: variant  <dig>   <cit>  and variant  <dig>   <cit> , both encoding the same alk <dig> protein of  <dig> amino acids.
mutations in acvrl <dig> give rise to an autosomal vascular dysplasia called hereditary haemorrhagic telangiectasia type  <dig>   <cit> , while hht type  <dig> is caused by mutations in eng, the gene coding for the tgf-β co-receptor endoglin  <cit> .
hht <dig> and hht <dig> are genetically dominant and mutant homozygosis is lethal, as confirmed in knock out mice for eng and acvrl <dig>  <cit> .
current estimates suggest that one in  <dig> - <dig>  people are affected by hht  <cit> .
patients are clinically diagnosed according to the curaçao criteria  <cit> , including nose bleeds, mucocutaneous telangiectases, internal arteriovenous malformations and familial inheritance.
around 90% of hht patients have been genetically diagnosed as hht <dig> or hht <dig>  and multiple mutations along exonic regions of acvrl <dig> and eng have been described  <cit> .
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  <cit>  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  <cit> , very little has been done in relation to the acvrl <dig> promoter.
in this work, we have analyzed the 5'-proximal acvrl <dig> promoter region, characterized new transcriptional initiation points, and analyzed the transcriptional mechanisms that regulate its expression.
RESULTS
identification of novel tsss for human acvrl <dig> gene revealed by 5'race
in order to characterize the origins of acvrl <dig> transcription in endothelial cells, a 5'race of total rna obtained from huvecs was performed.
after nested pcr, the amplified bands were cloned and sequenced .
the three major products amplified correspond to three different 5'flanking regions.
more than 50% of the clones corresponded to the mrna <dig> of acvrl <dig> previously described  <cit> , whereas two new tsss  were also identified in several clones from independent experiments .
interestingly, both alternative mrnas begin the transcription upstream the tss , in the positions - <dig> and - <dig>  respectively.
accordingly, the exons have been renumbered in this work in such a way that the new exon  <dig> is upstream of the previously described tss .
the numbering of the tss  has been maintained because it appears to be the predominant tss.
thus, the previously considered exon  <dig> turns into exon  <dig>  being named 2a, in the case of nm_ <dig>  and 2b in the case of nm_ <dig>  likewise, the newly discovered upstream exon now becomes exon  <dig>  named 1a in the mrna <dig>  or 1b, in the variant mrna <dig>
the fragments obtained in the present work correspond to isoforms mrna <dig> and mrna <dig>  the corresponding cdna sequences have been deposited in genbank database with accession numbers hm <dig> and hm <dig>  respectively.
both of them reveal a cryptic exon placed upstream the +  <dig>  resulting in two, instead of one, transcribed but not translated exons.
this event gives rise to a rearrangement from  <dig> to  <dig> exons in the composition of the coding region of acvrl <dig>
the human acvrl <dig> promoter lacks tata/caat boxes and has multiple sp <dig> motifs
human acvrl <dig> gene resides on chromosome 12q11-q <dig>  <cit> .
thus, a  <dig>  bp fragment comprising from position - <dig> , <dig> to position - <dig> , <dig> of the contig  was amplified by pcr from human genomic dna .
this fragment corresponds to positions from - <dig>  bp to +  <dig> bp relative to the  tss.
interestingly, this 5'-proximal regulatory region of acvrl <dig> lacks tata and caat boxes.
a predictive in silico analysis of acvrl <dig> promoter using the genomatix matinspector software tool revealed a very high number of gc-rich regions and multiple putative sp <dig> binding consensus sites .
fourteen different possible sites for sp <dig> interaction were found, located in both strands: forward strand ; and reverse strand .
all of these sites have a very high similarity with the theoretical matrix .
three ap <dig> and four ap <dig> sites were found dispersed along the sequence.
in addition, the 5'-flanking sequence of acvrl <dig> gene contains several putative regulatory elements also present in other endothelial genes .
these consensus elements include eight motifs for the ets  family of transcription factors,  <dig> sequences sensitive for klf  recognition, seven nfκb  motifs, five e2f  consensus sequences, one smad binding element  and seven sites for rxr  dimers.
moreover, three hif  consensus sequences were found distributed along the acvrl <dig> promoter sequence.
note: start/end pos, starting/ending position of the consensus site in the sequence; str, strand-sense; matrix sim, matrix  similarity factor ; core sim, core consensus sequence  similarity factor ; ap <dig>  activator protein 1; ap <dig>  activator protein 4; cebp, ccaat/enhancer binding protein; eklf, erythroid krüppel-like factor; ets, e26-transformation-specific transcription factor; e2f, elongation factor 2; gklf, gut-related krüppel-like factor; hif, hypoxia inducible factor; nf?b, nuclear factor of kappa light polypeptide gene enhancer in b-cells; rxr, retinoid x receptor; smad, sma and mad-related; sp <dig>  specificity protein 1; zf <dig>  krüppel-like zinc finger protein  <dig>
the acvrl <dig> promoter region is highly conserved among different species
the acvrl <dig> proximal promoter sequence has several features that suggest a high level of regulation.
one of these features is the possibility to begin the transcription in different points.
to evaluate the importance of each putative regulatory site, the acvrl <dig> promoter of several mammalian species were compared using the clustalw <dig> algorithm.
the species analyzed included mouse, rat, cow, orangutan, rhesus monkey, horse, chimpanzee and dog.
for the alignments, we selected the same region than the one we were studying in humans .
figure  <dig> shows the comparative alignment of the acvrl <dig> human sequence surrounding the different tsss with the same regions in other animal species.
panel a shows a scheme of the regions aligned in panel b. the previously described tss  has a highly conserved sequence among primates , suggesting an important functional role.
moreover, the two newly described tsss are conserved only in this group of species .
of note, both sequences at the beginning of exons 1a and 1b are less conserved than the region surrounding tss .
another interesting feature about the putative acvrl <dig> promoter is the high number of sp <dig> consensus sites along the sequence, and near to the tsss.
these sites are important as possible key regulators for acvrl <dig> transcription, so we compared these motifs among different species.
the scheme of sp <dig> sites present in the sequence is shown in figure 4a.
the alignments of these fragments and the same regions from other animal species are included in figure 4b.
of note, these sites appear highly conserved, especially between humans and other primates, which is a characteristic of promoters under sp <dig> regulation and lacking tata/caat boxes.
basal activity of acvrl <dig> promoter suggests the presence of possible positive and negative regulatory regions
to assess the transcriptional activity of the acvrl <dig> promoter fragment, it was cloned into a luciferase reporter vector.
a schematic representation of the acvrl <dig> promoter fragment cloned into pgl2-luc, with the locations of the different tss and the atg translation start codon, is shown in figure 5a.
serial 5'-deleted constructs from - <dig>  bp to - <dig> bp of the acvrl <dig> proximal promoter were generated  and their basal transcription activity assessed in hmec- <dig>  in this endothelial cell line, the shortest construct maintains the majority of the transcriptional activity, whereas the -422/+ <dig> construct displays one third of the activity compared with the whole sequence .
in order to unravel if acvrl <dig> could be transcriptionally regulated by its own ligands, the effect of tgf-β <dig> or bmp <dig> was assessed.
hmec- <dig> cells were transfected with different palk <dig> reporter constructs and treated with either tgf-β <dig> or bmp <dig> using the time and dose previously established in endothelial cells  <cit> .
none of these treatments had a significant effect on the transcriptional activity of the reporter constructs, except for the construct -422/+ <dig>  whose basal activity seems to be slightly upregulated in the presence of bmp <dig> .
sp <dig> is critical for the acvrl <dig> basal transcription
since  <dig> putative target sequences for sp <dig> were predicted in the acvrl <dig> promoter, their contribution to the basal activity was assessed in schneider drosophila embryonic s <dig> cells.
these cells are undifferentiated and do not express endogenous sp <dig> or any other related member of its family.
as shown in figure 6a, left, the proximal promoter of acvrl <dig> shows background levels of basal activity in the absence of sp <dig>  by contrast, ectopic expression of sp <dig> shows a dramatic effect on palk <dig> activity.
even with very low amounts of the transfected sp <dig> expression vector, the basal transcription of palk <dig> is increased ~  <dig> fold following a dose-response curve.
there is a saturating effect around  <dig> ng sp <dig> where the promoter is stimulated ~ <dig> fold.
the same experiment performed in hek293t cells resulted in the stimulation of the basal activity up to 3-fold .
this moderate increase, as compared to s <dig> cells , is likely due to the endogenous expression of sp <dig> in hek293t cells.
thus, small amounts of sp <dig> expression are sufficient to saturate all the sp <dig> sites in the acvrl <dig> promoter, leading to the maximum transcriptional activity that the promoter can achieve.
the next step was to dissect the sp <dig> transcription induction in the different promoter constructs.
s <dig> cells were co-transfected with sp <dig> and with the different 5'-deleted constructs of the reporter palk <dig> .
all constructs showed low basal transcriptional activities, which have been referred to value  <dig> and the stimulation is expressed as fold induction.
when as little as  <dig> ng of the sp <dig> expression vector was transfected, the activity levels of each construct were remarkably stimulated .
it is worth mentioning that the maximum activity reached by the -422/+ <dig> construct did not parallel its relatively low activity in endothelial cells, which have basal sp <dig> levels .
this discrepancy is probably explained by the different background of transcription factors present in drosophila embryonic versus human hmec- <dig> cells.
to further assess the relevance of sp <dig> in acvrl <dig> transcription, sp <dig> expression was abolished in mammalian sp1-expressing cells.
thus, hek293t cells transfected with sirna targeted to sp <dig> led to a marked decrease  of the acvrl <dig> transcriptional activity in all the constructs .
these data confirm that sp <dig> is essential for the transcriptional activity of the acvrl <dig> promoter.
chromatin immunoprecipitation of endogenous sp <dig> bound to acvrl <dig> promoter in huvecs
because sp <dig> is clearly able to stimulate the basal transcription of acvrl <dig> in vitro, it was of interest to assess whether the physical binding of sp <dig> to the acvrl <dig> promoter occurs within the endothelial cells.
for this purpose, a chromatin immunoprecipitation  of endogenous sp <dig> was performed in huvecs.
four different regions were screened, spanning the whole - <dig> /+ <dig> bp sequence of acvrl <dig> promoter .
pcr amplification yielded positive bands along the four fragments, suggesting that sp <dig> is normally bound to all these fragments of the acvrl <dig> promoter and, consequently, most of the putative theoretical binding sites are functionally active .
densitometric analysis of the pcr enriched fragments revealed that all of them showed a clear sp <dig> binding above background, yielding the strongest signal the fragment -510/- <dig> .
as a negative control, the erythropoietin  promoter, containing a sp <dig> motif that recruits sp <dig> only under hypoxic conditions  <cit> , was used.
in fact, the chip experiment demonstrated that sp <dig> did not bind to this sp <dig> site in the epo promoter, confirming the specificity of the assay .
considering that the smallest fragment  maintains the majority of the promoter activity respect to the whole construct , the nearest sp <dig> sites upstream of the tss , appear to be critical for the acvrl <dig> basal transcription.
interestingly, the -89/- <dig> fragment encompasses two adjacent sp <dig> consensus elements, flanking a putative klf <dig> binding site .
this fragment was selected as a probe for emsa studies.
a mobility shift appeared when nuclear extracts from hela cells  were incubated with the labelled probe and this binding was effectively competed with 100-fold excess of cold probe .
a supershift was observed in the presence of an antibody specific for sp <dig> whereas anti-sp <dig> or anti-nfκb antibodies did not affect the mobility shift, demonstrating the specificity for sp <dig>  the slight decrease of the retarded band in the presence of anti-sp <dig> may be explained by the fact that sp <dig> and sp <dig> often bind to the same sites.
furthermore, single mutations of either sp <dig> sites yielded retarded bands of a weaker intensity, suggesting that both sites are binding sp <dig> independently.
as negative controls, two sp <dig> unrelated sequences, -823/- <dig>  and aatt  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 alk <dig> expression in endothelial cells
acvrl <dig> promoter showed several gc-rich regions in the in silico analysis of the - <dig> /+ <dig> bp fragment.
using cpgplot software, two cpg islands were identified, based on their parameters: 1) cg percentage > 50%; and 2) a ratio of observed-to-expected >  <dig> .
these two islands comprise the regions between -408/- <dig> and -177/+ <dig> .
because these gc-rich regions are potential targets for sp <dig> binding and methylation of cytosines in cpg islands is one of the main epigenetic modifications that regulate gene expression, we analyzed whether alk <dig> expression could be influenced by promoter methylation.
indeed, as observed by real time pcr, when endothelial hmec- <dig> and huvec cells were subjected to a treatment with the demethylating agent 5'-aza-2'-deoxycytidine , their alk <dig> levels were upregulated up to ~ <dig> -fold .
interestingly, hek293t, a cell line that does not express alk <dig> under normal conditions , underwent ~ <dig> -fold activation of alk <dig> expression after treatment with 5-aza-dc .
likewise, id <dig>  which is a specific target gene of the alk1/tgf-β <dig> signaling cascade activation, became upregulated in parallel to the increased levels of alk <dig> in the three cell types .
moreover, the activity of the reporter p2-luc, specific of the alk <dig> signalling pathway, was strongly enhanced upon treatment with 5-aza-dc.
conversely, the effect of in vitro hypermethylation was assessed.
acvrl <dig> promoter constructs were methylated in vitro with the cpg methyltransferase m.sssi and the methylation status was checked by comparative digestion of both mock and methylated acvrl <dig> promoter construct with hpaii .
then, hek293t cells were transfected with mock-methylated or hypermethylated promoter constructs.
as shown in figure 9a the transcriptional activity of all the acvrl <dig> constructs was completely abolished upon methylation.
by contrast, the activity of the prolactin promoter region driven by a tata box was almost unaffected , whereas the activity of id <dig> promoter construct was reduced at a much lower degree than the acvrl <dig> construct  .
to assess whether the methylation status of the sp <dig> motifs affected the sp <dig> binding, the -89/- <dig> radiolabelled probe was competed with cold unmethylated and methylated probes.
as shown in figure 9c, the unmethylated probe clearly inhibited the sp <dig> band, whereas the methylated probe was unable to compete.
these results suggest that the methylation status of the promoter strongly modulates alk <dig> expression.
discussion
the characterization of the 5'-proximal acvrl <dig> 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 acvrl <dig> may have several tsss.
thus, acvrl <dig>  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 acvrl <dig> 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  <cit> .
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 acvrl <dig>  the existence of different transcribed untranslated regions suggests regulation of alk <dig> 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 acvrl <dig> transcripts described in human, orangutan, rhesus monkey and chimpanzee, seem to begin with an untranslated first exon.
thus, acvrl <dig> 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  displays a similar activity to that of the whole fragment .
this finding suggests the existence of critical transcription factors binding to this area in order to enhance the transcriptional activity of the tata-less acvrl <dig> promoter.
in agreement with this view, a high degree of conservation around the +  <dig> tss among different species was found.
the analysis also suggests the presence of a putative "repressor" segment between - <dig> and - <dig> positions, which inhibits acvrl <dig> transcription.
interestingly, a similar region is found in the eng promoter further upstream  <cit> , something that deserves further investigation.
the in silico study of the acvrl <dig> promoter sequence showed several important putative consensus elements, also found in eng promoter  <cit>  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, acvrl <dig> may be regulated by some of the transcription factors also controlling eng expression.
in this sense, because eng is regulated by hif- <dig>  smad, klf <dig>  ets, or sp <dig>  <cit> , it will be of interest to assess their functional implications in acvrl <dig>  of note, three sequences matching hif-1α motifs were found at positions -808/- <dig>  -286/- <dig> and -236/- <dig> bp.
the possibility that acvrl <dig> could respond to hif-1α is supported because hypoxia promotes angiogenesis, a process that is regulated by alk <dig>  in addition, multiple klf consensus elements are dispersed along the acvrl <dig> promoter region.
interestingly, this is another feature owing to the tgf-β receptor complex genes, in which the transcription is stimulated by klf <dig> as an early injury response  <cit> .
on the other hand, the presence of multiple ets sites, suggests regulation by mapks   <cit> .
in this sense, in the functionally related gene, eng, an ets functional site was described in the proximal promoter  <cit> .
we also note that the presence of ap <dig> and nfκb sites suggests that acvrl <dig> 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 - <dig> bp position of the acvrl <dig> promoter.
also, at variance with eng, the acvrl <dig> promoter does not contain any tgf-β-responsive element  in the neighbourhood of sp <dig> sites.
moreover, the tgf-β receptor type i  gene , contains a sbe within the proximal promoter and both eng and tgfbri mrna expression is upregulated by tgf-β  <cit> .
thus, the proximal region of eng is regulated by tgf-β through smads, and a direct cooperation smad/sp <dig> giving rise to an important increase in transcription  <cit> .
consistent with the lack of proximal responsive tgf-β elements within the acvrl <dig> promoter, we found that its promoter activity was not regulated by tgf-β <dig> or bmp <dig>  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 alk <dig> to activate signalling through smads.
addressing the functionality of all these transcription factors in the acvrl <dig> promoter deserves an independent investigation.
the present work has focused on the functional role of sp <dig> on acvrl <dig> basal transcription.
the most striking feature revealed by the in silico analysis of acvrl <dig> is the presence of multiple sp <dig> sites next to the different tsss.
sp <dig> 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  <cit> .
interestingly, this is also the case of several tgf-β receptors such as eng and tgfbr <dig>  whose transcription is driven by sp <dig>  <cit> .
further support for the involvement of sp <dig> was obtained from the highly conserved alignment of the sp <dig> consensus sites along the acvrl <dig> promoter among several species that suggests a conserved biological relevance of sp <dig> in the transcription of acvrl <dig>
we have demonstrated here that sp <dig> is a key factor necessary for the basal activation of acvrl <dig> transcription.
this effect has been observed both by sp <dig> overexpression in cells lacking endogenous sp <dig>  and by interfering endogenous sp <dig> with sirna in mammalian cells.
the large number of sp <dig> consensus sites found in the acvrl <dig> promoter, and the finding that small amounts of sp <dig> can saturate its transcription, points out to a critical dependence on sp <dig>  and a fine tuning of acvrl <dig> transcription by sp <dig>  furthermore, a chip analysis in huvecs after chromatin crosslinking revealed that sp <dig> binds in vivo to the different consensus motifs along the whole proximal acvrl <dig> promoter sequence studied .
thus, in endothelial primary cells growing in a rich medium, sp <dig> may be binding to most of the specific motifs within the acvrl <dig> promoter region.
it can be speculated that the sp <dig> binding to the starting transcriptional machinery complex is needed to ensure a transcription driven by sp <dig> using different tsss.
because the closest region to the +  <dig> tss is framed by the construction -284/+ <dig> palk <dig> and its transcriptional levels are similar to those of the - <dig> /+ <dig> palk <dig> vector, probably the functional involvement of sp <dig> for acvrl <dig> transcription initiation is critical in this region.
when searching the sp <dig> consensus sites within this region, an interesting double site in the -89/- <dig> region was found.
this sp <dig> rich fragment contains two putative sp <dig> consensus sites  and, as shown in emsa experiments, both sites are functional in binding to sp <dig>
one key factor that modulates the sp <dig> transcription dependency is the degree of methylation of the cpg islands contained within the sp <dig> consensus elements.
this is consistent with the observation that dna methylation may interfere with the binding of sp <dig> to dna  <cit> .
in fact, we found that methylation of the -89/- <dig> sp <dig> probe of acvrl <dig> rendered this consensus motif inactive for sp <dig> binding.
it is well established that dna methylation of cpg islands is an important mechanism for transcriptional regulation of multiple genes in mammals  <cit> .
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  <cit> .
in some genes, cytosine methylation of sp <dig> sites has been reported as a marker of organ-specific expression and as a specific regulator of the expression  <cit> .
that could be the case of acvrl <dig>  in which demethylation of dna in endothelial cells leads to a marked increase of acvrl <dig> transcription, potentially because demethylation of cpg islands within the sp <dig> sites is involved in acvrl <dig> basal transcription.
alk <dig> expression has been reported in several cell types, but its major roles are related to its predominant expression in endothelium.
the alk <dig> 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  <cit> .
the endothelial specific expression may be explained by the presence within the proximal promoter region of acvrl <dig> of consensus motifs for transcription factors  also shared by other endothelial specific genes such as eng, pecam <dig> , vegfr <dig> , cdh <dig> , enos/nos- <dig>  or tie <dig>   <cit> .
in addition, the involvement of distal regulatory regions in the human acvrl <dig>  such as the one described in mouse that confers arterial endothelium-specificity, can not be excluded  <cit> .
whether the degree of acvrl <dig> methylation correlates with the endothelial specific expression of alk <dig>  remains to be explored.
CONCLUSIONS
novel acvrl <dig> transcripts have been identified, the acvrl <dig> promoter has been characterized and its regulation by sp <dig> has been demonstrated.
furthermore, a close dependence between acvrl <dig> expression and the cg methylation degree was found.
future experiments to identify other trans-acting or trans-repressing factors in acvrl <dig> regulation remain to be addressed.

 alk is a transmembrane type i receptor of the transforming growth factor-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, 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. alk is involved in angiogenesis, and there is growing evidence indicating that it plays a key function in the arterial/venous differentiation during embryonic vascular development. it has been reported that alk interacts with three ligands: with tgf-β and tgf-β in complex with the receptor type ii; and with bone morphogenetic protein, in complex with the activin receptor type iia or the bmp receptor type ii. 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, receptor type i and an ancillary co-receptor. alk is the predominant tβr-i in the majority of the cell types, but in endothelial cells alk shares the tβr-i function with alk in vitro. the significance of this apparent redundancy is explained because alk and alk signal in opposite directions, balancing the tgf-β signalling pathway in this cell type. alk 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. alk 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. alk signals through smad2/ to regulate pai-, collagen i, or nos-3/enos, whereas alk signals through smad1/5/ to induce genes involved in proliferation such as id, id, smad smad eng or bmprii. in addition, it has been shown that bmp is a quiescence factor for the microvasculature. the gene encoding alk spans bp within the large arm of chromosome, coded on the positive strand. acvrl cdna was described first in simultaneously by two different groups showing a characteristic transcriptional start site, where the first exon is transcribed but not translated. to date, two different variants of mrna transcripts have been described: variant and variant, both encoding the same alk protein of amino acids. mutations in acvrl give rise to an autosomal vascular dysplasia called hereditary haemorrhagic telangiectasia type, while hht type is caused by mutations in eng, the gene coding for the tgf-β co-receptor endoglin. hht and hht are genetically dominant and mutant homozygosis is lethal, as confirmed in knock out mice for eng and acvrl. current estimates suggest that one in - 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 hht or hht and multiple mutations along exonic regions of acvrl 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, very little has been done in relation to the acvrl promoter. in this work, we have analyzed the 5'-proximal acvrl promoter region, characterized new transcriptional initiation points, and analyzed the transcriptional mechanisms that regulate its expression.  identification of novel tsss for human acvrl gene revealed by 5'race in order to characterize the origins of acvrl transcription in endothelial cells, a 5'race of total rna obtained from huvecs was performed. after nested pcr, the amplified bands were cloned and sequenced. the three major products amplified correspond to three different 5'flanking regions. more than 50% of the clones corresponded to the mrna of acvrl previously described, whereas two new tsss were also identified in several clones from independent experiments. interestingly, both alternative mrnas begin the transcription upstream the tss, in the positions - and - respectively. accordingly, the exons have been renumbered in this work in such a way that the new exon is upstream of the previously described tss. the numbering of the tss has been maintained because it appears to be the predominant tss. thus, the previously considered exon turns into exon being named 2a, in the case of nm_ and 2b in the case of nm_ likewise, the newly discovered upstream exon now becomes exon named 1a in the mrna or 1b, in the variant mrna the fragments obtained in the present work correspond to isoforms mrna and mrna the corresponding cdna sequences have been deposited in genbank database with accession numbers hm and hm respectively. both of them reveal a cryptic exon placed upstream the + resulting in two, instead of one, transcribed but not translated exons. this event gives rise to a rearrangement from to exons in the composition of the coding region of acvrl the human acvrl promoter lacks tata/caat boxes and has multiple sp motifs human acvrl gene resides on chromosome 12q11-q. thus, a bp fragment comprising from position -, to position -, of the contig was amplified by pcr from human genomic dna. this fragment corresponds to positions from - bp to + bp relative to the tss. interestingly, this 5'-proximal regulatory region of acvrl lacks tata and caat boxes. a predictive in silico analysis of acvrl promoter using the genomatix matinspector software tool revealed a very high number of gc-rich regions and multiple putative sp binding consensus sites. fourteen different possible sites for sp interaction were found, located in both strands: forward strand; and reverse strand. all of these sites have a very high similarity with the theoretical matrix. three ap and four ap sites were found dispersed along the sequence. in addition, the 5'-flanking sequence of acvrl gene contains several putative regulatory elements also present in other endothelial genes. these consensus elements include eight motifs for the ets family of transcription factors, sequences sensitive for klf recognition, seven nfκb motifs, five e2f consensus sequences, one smad binding element and seven sites for rxr dimers. moreover, three hif consensus sequences were found distributed along the acvrl promoter sequence. note: start/end pos, starting/ending position of the consensus site in the sequence; str, strand-sense; matrix sim, matrix similarity factor; core sim, core consensus sequence similarity factor; ap activator protein 1; ap activator protein 4; cebp, ccaat/enhancer binding protein; eklf, erythroid krüppel-like factor; ets, e26-transformation-specific transcription factor; e2f, elongation factor 2; gklf, gut-related krüppel-like factor; hif, hypoxia inducible factor; nf?b, nuclear factor of kappa light polypeptide gene enhancer in b-cells; rxr, retinoid x receptor; smad, sma and mad-related; sp specificity protein 1; zf krüppel-like zinc finger protein the acvrl promoter region is highly conserved among different species the acvrl proximal promoter sequence has several features that suggest a high level of regulation. one of these features is the possibility to begin the transcription in different points. to evaluate the importance of each putative regulatory site, the acvrl promoter of several mammalian species were compared using the clustalw algorithm. the species analyzed included mouse, rat, cow, orangutan, rhesus monkey, horse, chimpanzee and dog. for the alignments, we selected the same region than the one we were studying in humans. figure shows the comparative alignment of the acvrl human sequence surrounding the different tsss with the same regions in other animal species. panel a shows a scheme of the regions aligned in panel b. the previously described tss has a highly conserved sequence among primates, suggesting an important functional role. moreover, the two newly described tsss are conserved only in this group of species. of note, both sequences at the beginning of exons 1a and 1b are less conserved than the region surrounding tss. another interesting feature about the putative acvrl promoter is the high number of sp consensus sites along the sequence, and near to the tsss. these sites are important as possible key regulators for acvrl transcription, so we compared these motifs among different species. the scheme of sp sites present in the sequence is shown in figure 4a. the alignments of these fragments and the same regions from other animal species are included in figure 4b. of note, these sites appear highly conserved, especially between humans and other primates, which is a characteristic of promoters under sp regulation and lacking tata/caat boxes. basal activity of acvrl promoter suggests the presence of possible positive and negative regulatory regions to assess the transcriptional activity of the acvrl promoter fragment, it was cloned into a luciferase reporter vector. a schematic representation of the acvrl promoter fragment cloned into pgl2-luc, with the locations of the different tss and the atg translation start codon, is shown in figure 5a. serial 5'-deleted constructs from - bp to - bp of the acvrl proximal promoter were generated and their basal transcription activity assessed in hmec- in this endothelial cell line, the shortest construct maintains the majority of the transcriptional activity, whereas the -422/+ construct displays one third of the activity compared with the whole sequence. in order to unravel if acvrl could be transcriptionally regulated by its own ligands, the effect of tgf-β or bmp was assessed. hmec- cells were transfected with different palk reporter constructs and treated with either tgf-β or bmp using the time and dose previously established in endothelial cells. none of these treatments had a significant effect on the transcriptional activity of the reporter constructs, except for the construct -422/+ whose basal activity seems to be slightly upregulated in the presence of bmp. sp is critical for the acvrl basal transcription since putative target sequences for sp were predicted in the acvrl promoter, their contribution to the basal activity was assessed in schneider drosophila embryonic s cells. these cells are undifferentiated and do not express endogenous sp or any other related member of its family. as shown in figure 6a, left, the proximal promoter of acvrl shows background levels of basal activity in the absence of sp by contrast, ectopic expression of sp shows a dramatic effect on palk activity. even with very low amounts of the transfected sp expression vector, the basal transcription of palk is increased ~ fold following a dose-response curve. there is a saturating effect around ng sp where the promoter is stimulated ~ fold. the same experiment performed in hek293t cells resulted in the stimulation of the basal activity up to 3-fold. this moderate increase, as compared to s cells, is likely due to the endogenous expression of sp in hek293t cells. thus, small amounts of sp expression are sufficient to saturate all the sp sites in the acvrl promoter, leading to the maximum transcriptional activity that the promoter can achieve. the next step was to dissect the sp transcription induction in the different promoter constructs. s cells were co-transfected with sp and with the different 5'-deleted constructs of the reporter palk. all constructs showed low basal transcriptional activities, which have been referred to value and the stimulation is expressed as fold induction. when as little as ng of the sp expression vector was transfected, the activity levels of each construct were remarkably stimulated. it is worth mentioning that the maximum activity reached by the -422/+ construct did not parallel its relatively low activity in endothelial cells, which have basal sp levels. this discrepancy is probably explained by the different background of transcription factors present in drosophila embryonic versus human hmec- cells. to further assess the relevance of sp in acvrl transcription, sp expression was abolished in mammalian sp1-expressing cells. thus, hek293t cells transfected with sirna targeted to sp led to a marked decrease of the acvrl transcriptional activity in all the constructs. these data confirm that sp is essential for the transcriptional activity of the acvrl promoter. chromatin immunoprecipitation of endogenous sp bound to acvrl promoter in huvecs because sp is clearly able to stimulate the basal transcription of acvrl in vitro, it was of interest to assess whether the physical binding of sp to the acvrl promoter occurs within the endothelial cells. for this purpose, a chromatin immunoprecipitation of endogenous sp was performed in huvecs. four different regions were screened, spanning the whole - /+ bp sequence of acvrl promoter. pcr amplification yielded positive bands along the four fragments, suggesting that sp is normally bound to all these fragments of the acvrl promoter and, consequently, most of the putative theoretical binding sites are functionally active. densitometric analysis of the pcr enriched fragments revealed that all of them showed a clear sp binding above background, yielding the strongest signal the fragment -510/-. as a negative control, the erythropoietin promoter, containing a sp motif that recruits sp only under hypoxic conditions, was used. in fact, the chip experiment demonstrated that sp did not bind to this sp site in the epo promoter, confirming the specificity of the assay. considering that the smallest fragment maintains the majority of the promoter activity respect to the whole construct, the nearest sp sites upstream of the tss, appear to be critical for the acvrl basal transcription. interestingly, the -89/- fragment encompasses two adjacent sp consensus elements, flanking a putative klf binding site. this fragment was selected as a probe for emsa studies. a mobility shift appeared when nuclear extracts from hela cells were incubated with the labelled probe and this binding was effectively competed with 100-fold excess of cold probe. a supershift was observed in the presence of an antibody specific for sp whereas anti-sp or anti-nfκb antibodies did not affect the mobility shift, demonstrating the specificity for sp the slight decrease of the retarded band in the presence of anti-sp may be explained by the fact that sp and sp often bind to the same sites. furthermore, single mutations of either sp sites yielded retarded bands of a weaker intensity, suggesting that both sites are binding sp independently. as negative controls, two sp unrelated sequences, -823/- and aatt 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 alk expression in endothelial cells acvrl promoter showed several gc-rich regions in the in silico analysis of the - /+ bp fragment. using cpgplot software, two cpg islands were identified, based on their parameters: 1) cg percentage > 50%; and 2) a ratio of observed-to-expected >. these two islands comprise the regions between -408/- and -177/+. because these gc-rich regions are potential targets for sp binding and methylation of cytosines in cpg islands is one of the main epigenetic modifications that regulate gene expression, we analyzed whether alk expression could be influenced by promoter methylation. indeed, as observed by real time pcr, when endothelial hmec- and huvec cells were subjected to a treatment with the demethylating agent 5'-aza-2'-deoxycytidine, their alk levels were upregulated up to ~ -fold. interestingly, hek293t, a cell line that does not express alk under normal conditions, underwent ~ -fold activation of alk expression after treatment with 5-aza-dc. likewise, id which is a specific target gene of the alk1/tgf-β signaling cascade activation, became upregulated in parallel to the increased levels of alk in the three cell types. moreover, the activity of the reporter p2-luc, specific of the alk signalling pathway, was strongly enhanced upon treatment with 5-aza-dc. conversely, the effect of in vitro hypermethylation was assessed. acvrl promoter constructs were methylated in vitro with the cpg methyltransferase m.sssi and the methylation status was checked by comparative digestion of both mock and methylated acvrl promoter construct with hpaii. then, hek293t cells were transfected with mock-methylated or hypermethylated promoter constructs. as shown in figure 9a the transcriptional activity of all the acvrl constructs was completely abolished upon methylation. by contrast, the activity of the prolactin promoter region driven by a tata box was almost unaffected, whereas the activity of id promoter construct was reduced at a much lower degree than the acvrl construct. to assess whether the methylation status of the sp motifs affected the sp binding, the -89/- radiolabelled probe was competed with cold unmethylated and methylated probes. as shown in figure 9c, the unmethylated probe clearly inhibited the sp band, whereas the methylated probe was unable to compete. these results suggest that the methylation status of the promoter strongly modulates alk expression. discussion the characterization of the 5'-proximal acvrl 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 acvrl may have several tsss. thus, acvrl 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 acvrl 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. 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 acvrl the existence of different transcribed untranslated regions suggests regulation of alk 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 acvrl transcripts described in human, orangutan, rhesus monkey and chimpanzee, seem to begin with an untranslated first exon. thus, acvrl 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 displays a similar activity to that of the whole fragment. this finding suggests the existence of critical transcription factors binding to this area in order to enhance the transcriptional activity of the tata-less acvrl promoter. in agreement with this view, a high degree of conservation around the + tss among different species was found. the analysis also suggests the presence of a putative "repressor" segment between - and - positions, which inhibits acvrl transcription. interestingly, a similar region is found in the eng promoter further upstream, something that deserves further investigation. the in silico study of the acvrl promoter sequence showed several important putative consensus elements, also found in eng promoter 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, acvrl may be regulated by some of the transcription factors also controlling eng expression. in this sense, because eng is regulated by hif- smad, klf ets, or sp, it will be of interest to assess their functional implications in acvrl of note, three sequences matching hif-1α motifs were found at positions -808/- -286/- and -236/- bp. the possibility that acvrl could respond to hif-1α is supported because hypoxia promotes angiogenesis, a process that is regulated by alk in addition, multiple klf consensus elements are dispersed along the acvrl promoter region. interestingly, this is another feature owing to the tgf-β receptor complex genes, in which the transcription is stimulated by klf as an early injury response. on the other hand, the presence of multiple ets sites, suggests regulation by mapks. 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 ap and nfκb sites suggests that acvrl 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 - bp position of the acvrl promoter. also, at variance with eng, the acvrl promoter does not contain any tgf-β-responsive element in the neighbourhood of sp sites. moreover, the tgf-β receptor type i gene, contains a sbe within the proximal promoter and both eng and tgfbri mrna expression is upregulated by tgf-β. thus, the proximal region of eng is regulated by tgf-β through smads, and a direct cooperation smad/sp giving rise to an important increase in transcription. consistent with the lack of proximal responsive tgf-β elements within the acvrl promoter, we found that its promoter activity was not regulated by tgf-β or bmp 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 alk to activate signalling through smads. addressing the functionality of all these transcription factors in the acvrl promoter deserves an independent investigation. the present work has focused on the functional role of sp on acvrl basal transcription. the most striking feature revealed by the in silico analysis of acvrl is the presence of multiple sp sites next to the different tsss. sp 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 tgfbr whose transcription is driven by sp. further support for the involvement of sp was obtained from the highly conserved alignment of the sp consensus sites along the acvrl promoter among several species that suggests a conserved biological relevance of sp in the transcription of acvrl we have demonstrated here that sp is a key factor necessary for the basal activation of acvrl transcription. this effect has been observed both by sp overexpression in cells lacking endogenous sp and by interfering endogenous sp with sirna in mammalian cells. the large number of sp consensus sites found in the acvrl promoter, and the finding that small amounts of sp can saturate its transcription, points out to a critical dependence on sp and a fine tuning of acvrl transcription by sp furthermore, a chip analysis in huvecs after chromatin crosslinking revealed that sp binds in vivo to the different consensus motifs along the whole proximal acvrl promoter sequence studied. thus, in endothelial primary cells growing in a rich medium, sp may be binding to most of the specific motifs within the acvrl promoter region. it can be speculated that the sp binding to the starting transcriptional machinery complex is needed to ensure a transcription driven by sp using different tsss. because the closest region to the + tss is framed by the construction -284/+ palk and its transcriptional levels are similar to those of the - /+ palk vector, probably the functional involvement of sp for acvrl transcription initiation is critical in this region. when searching the sp consensus sites within this region, an interesting double site in the -89/- region was found. this sp rich fragment contains two putative sp consensus sites and, as shown in emsa experiments, both sites are functional in binding to sp one key factor that modulates the sp transcription dependency is the degree of methylation of the cpg islands contained within the sp consensus elements. this is consistent with the observation that dna methylation may interfere with the binding of sp to dna. in fact, we found that methylation of the -89/- sp probe of acvrl rendered this consensus motif inactive for sp 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 sp 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 acvrl in which demethylation of dna in endothelial cells leads to a marked increase of acvrl transcription, potentially because demethylation of cpg islands within the sp sites is involved in acvrl basal transcription. alk expression has been reported in several cell types, but its major roles are related to its predominant expression in endothelium. the alk 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 acvrl of consensus motifs for transcription factors also shared by other endothelial specific genes such as eng, pecam, vegfr, cdh, enos/nos- or tie. in addition, the involvement of distal regulatory regions in the human acvrl such as the one described in mouse that confers arterial endothelium-specificity, can not be excluded. whether the degree of acvrl methylation correlates with the endothelial specific expression of alk remains to be explored.  novel acvrl transcripts have been identified, the acvrl promoter has been characterized and its regulation by sp has been demonstrated. furthermore, a close dependence between acvrl expression and the cg methylation degree was found. future experiments to identify other trans-acting or trans-repressing factors in acvrl regulation remain to be addressed. 
moreover, silencing sp in hek293t cells resulted in a marked decrease of acvrl transcriptional activity. the 5'-proximal promoter region of acvrl was analyzed in silico, finding that it lacks tata/caat boxes, but contains a remarkably high number of gc-rich sp consensus sites. chromatin immunoprecipitation assays demonstrated multiple sp binding sites along the proximal promoter region of acvrl in endothelial cells. furthermore, demethylation of cpg islands, led to an increase in acvrl transcription, whereas in vitro hypermethylation resulted in the abolishment of sp1-dependent transcriptional activation of acvrl moreover, our data show that the methylation status of cpg islands markedly modulates the sp regulation of acvrl gene transcriptional activity. in spite of its patho-physiological relevance, little is known about the transcriptional regulation of acvrl here, we have studied the different origins of acvrl transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of sp in the transcriptional regulation of acvrl our results describe two new transcriptional start sites in acvrl gene, and indicate that sp is a key regulator of acvrl transcription, providing new insights into the molecular mechanisms that contribute to the expression of acvrl gene. activin receptor-like kinase is a transforming growth factor-β receptor type i, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis.
 activin receptor-like kinase is a transforming growth factor-β receptor type i, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. mutations in the alk gene 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 acvrl here, we have studied the different origins of acvrl transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of sp in the transcriptional regulation of acvrl  we have performed a 5'rapid amplification of cdna ends of acvrl 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 acvrl was analyzed in silico, finding that it lacks tata/caat boxes, but contains a remarkably high number of gc-rich sp consensus sites. in cells lacking sp acvrl promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of sp triggered a dose-dependent stimulation of its transcription. moreover, silencing sp in hek293t cells resulted in a marked decrease of acvrl transcriptional activity. chromatin immunoprecipitation assays demonstrated multiple sp binding sites along the proximal promoter region of acvrl in endothelial cells. furthermore, demethylation of cpg islands, led to an increase in acvrl transcription, whereas in vitro hypermethylation resulted in the abolishment of sp1-dependent transcriptional activation of acvrl  our results describe two new transcriptional start sites in acvrl gene, and indicate that sp is a key regulator of acvrl transcription, providing new insights into the molecular mechanisms that contribute to the expression of acvrl gene. moreover, our data show that the methylation status of cpg islands markedly modulates the sp regulation of acvrl gene transcriptional activity. 