BACKGROUND
the human bcl2l <dig> locus, located on chromosome 2q <dig>  encodes a protein of  <dig> amino acids structurally and functionally related to the bh3-only group of pro-apoptotic bcl <dig> family members  <cit> .
the gene is frequently mutated in diverse human tumours leading to loss of bcl2l <dig> activity  <cit> .
expression of bcl2l <dig> is induced by a diverse range of apoptotic stimuli such as deprivation of growth factors/cytokines, ionizing radiation, and cytotoxic peptides  <cit> .
although the regulation of bcl2l <dig> activity is highly complex and is cell context-dependent, studies aimed at identifying the molecular mechanisms underlying bcl2l <dig> activation during apoptosis have revealed an important role for the transcriptional regulation of bcl2l <dig> gene expression  <cit> .
a genomic region displaying promoter activity has been characterized for the human bcl2l <dig> locus  <cit> , while in the rat the existence of three alternative promoter sequences has been postulated, the most upstream of which corresponds to the promoter described for the human bcl2l <dig> gene  <cit> .
regulation of this conserved bcl2l <dig> promoter by foxo and e2f has been described using rat bcl2l <dig> genomic constructs, thus providing a likely mechanism for the induction of bcl2l <dig> expression during programmed cell death  <cit> , through the involvement of these transcription factors whose activity is induced in apoptotic contexts.
whether regulation by e2f and foxo factors is conserved in humans remains to be demonstrated; however, such studies are hampered by the relatively poor inter-specific sequence conservation in non-coding sequences and the paucity of information on the structure and regulation of the human bcl2l <dig> locus.
we therefore set out to investigate the existence of as yet uncharacterized human bcl2l <dig> promoter regions, and to determine the conservation from rodents to man of e2f regulation of bcl2l <dig> expression.
RESULTS
identification of a novel putative bcl2l <dig> promoter region and associated bcl2l <dig> exon
in order to further investigate genomic sequences of the human bcl2l <dig> locus with promoter activity, we first applied a bioinformatics approach to identify clusters of human ests with comparable 5' ends upstream of the first coding exon of the human bcl2l <dig> locus, informative of the possible existence of distinct transcript initiation sites.
we analyzed 13kb of human bcl2l <dig> genomic sequence immediately upstream of the first bcl2l <dig> coding exon by blast against the genbank human est database, and identified  <dig> clusters with identical/comparable 5'ends .
the largest group  corresponded well to the human bcl2l <dig> promoter already characterized , and conserved between human and rat bcl2l <dig> loci  <cit> .
a second, more heterogeneous group  localized to a region comprising sequences described in the rat as potentially harbouring  <dig> alternative promoters   <cit> .
a third group  localized to a region ca.
<dig>  kb upstream of the atg, which did not correspond to known bcl2l <dig> locus sequences in either man or rodents.
this group could be subdivided into two subgroups , each with identical or nearly identical 5' ends, apparently running in opposite directions and potentially suggestive of the existence of a bidirectional promoter.
the first indication of the likely existence of an as yet uncharacterized bcl2l <dig> promoter associated with novel bcl2l <dig> untranslated exon sequences came from the presence, within ests group  <dig>  of one est  which clearly comprised the first bcl2l <dig> coding exon , but not the first known bcl2l <dig> untranslated exon .
instead, novel untranslated exon sequences 5' of the first known untranslated bcl2l <dig> exon were present, which overlapped with group  <dig> subgroup b ests.
this was suggestive of the presence of a novel bcl2l <dig> promoter and associated untranslated exon  5' of the most upstream known bcl2l <dig> promoter .
we therefore focused on group  <dig> subgroup b ests, as these might represent novel bcl2l <dig> transcribed sequences by virtue of their overlapping with est db <dig>  which comprises the first bcl2l <dig> coding exon .
their experimental association with the first bcl2l <dig> coding exon  in a transcribed sequence would in fact confirm the existence of a new bcl2l <dig> promoter and associated untranslated exon.
therefore, a series of rt-pcr experiments were performed where the forward primer was anchored within group  <dig> subgroup b ests and the reverse primer was anchored within the first coding exon of the bcl2l <dig> locus, downstream of the translational initiation codon .
complementary dna  derived from hek <dig> mrna was used in these experiments, as these cells were shown to express bcl2l <dig> transcripts .
the sequence of the resulting amplicons  confirmed the presence of bcl2l <dig> coding sequences in the amplified cdna .
the alignment  of the obtained rt-pcr product sequences with db <dig> and with representative group  <dig>  subgroup b ests  confirmed the co-linearity of these transcribed sequences.
this finding confirmed that group  <dig>  subgroup b ests indeed comprise novel untranslated bcl2l <dig> sequences, thus identifying a novel bcl2l <dig> untranslated exon  and confirming the existence of at least one novel putative bcl2l <dig> promoter  upstream of the previously characterized human bcl2l <dig> promoter sequences .
having ascertained that group  <dig>  subgroup b ests correspond to bona fide bcl2l <dig> transcripts, we next sought to determine the 5' end of such transcripts in order to determine a putative transcript initiation site, defining a possible 5' exon boundary and a starting point for the definition of a promoter region for p <dig>  5'race experiments performed on hek <dig> cells, using a reverse primer anchored within the novel human bcl2l <dig> exon  and overlapping with the forward primer used in the rt-pcr studies described above, identified a sequence around the 5' end of group  <dig>  subgroup b ests as the transcript initiation site in hek <dig> cells , thus defining the likely 5' boundary of this novel human bcl2l <dig> exon and confirming the existence of a novel human bcl2l <dig> promoter.
a diagram summarizing the position of ests and of the rt-pcr and 5'race products relatively to human bcl2l <dig> genomic sequences is provided in fig.
4a.
as transcripts originating from this region do not contain sequences associated with the exon immediately downstream of the published human bcl2l <dig> promoter , but splice directly onto the first bcl2l <dig> coding exon , it follows that the human bcl2l <dig> locus might comprise at least two alternative promoters  and that alternative splicing occurs in the 5' untranslated exons.
a third promoter region  may also exist based on data from the rodent bcl2l <dig> locus  <cit> .
next, the capacity of p <dig> sequences to drive gene expression was tested in a series of reporter assays in hek <dig> cells.
a human genomic region of  <dig> bp region comprising a portion of exon  <dig>  was therefore amplified, cloned, sequenced and subcloned into the promoter-less luciferase reporter vector pgl3basic in both orientations, to give rise to the reporter constructs pbcl2l11-p1- <dig> and .
the constructs were then transiently transfected into hek <dig> cells and the resulting luciferase activity compared to basal pgl3basic activity .
the results  indicate that p <dig> sequences can drive gene expression, as measured by luciferase activity, though independently of their orientation.
although orientation-dependent activity is usually considered a hallmark of promoter sequences, examples of bona-fide bidirectional promoters are known .
therefore, p <dig> may represent a promoter with potential for bi-directional activity, at least in hek <dig> cells.
this possibility is also supported by the existence of group  <dig>  subgroup a ests as discussed above , representing ests with similar 5' ends but with apparently displaying sequences which are complementary to the sense  p <dig> direction and starting within ca.
<dig> bp of the transcript initiation site of e <dig>  although the existence of a bi-directional promoter in this genomic region is supported by our experimental data and from the existence of coherent ests, further studies  are required to confirm this possibility.
as our interest was on the identification of putative novel bcl2l <dig> promoter sequences, this line was not pursued further.
exon  <dig> containing transcripts are widely expressed in human tissues
in order to evaluate the possible significance of bcl2l <dig> transcripts originating from this novel candidate promoter for overall bcl2l <dig> transcript expression in man, the expression of bcl2l <dig> mrnas bearing the alternatively spliced exon  <dig> or exon  <dig> sequences, or of total bcl2l <dig> transcripts, was evaluated in a panel of cdnas derived from human tissues by real-time pcr, using sybr green technology.
primer pairs specific for bcl2l <dig> exon  <dig> or  <dig>  or for a bcl2l <dig> transcribed region presumably present in most characterized bcl2l <dig> transcripts  were employed.
the results indicated that transcripts originating from the candidate novel bcl2l <dig> promoter p <dig> were readily detectable in a number of human tissues , and contributed most significantly to bcl2l <dig> mrna levels in testis, heart and liver .
next, the relative contribution of exon 1- and exon 2-containing transcripts to total bcl2l <dig> expression in hek <dig> cells was then evaluated by real-time pcr, using sybr green technology.
the results  again indicated that bcl2l <dig> transcripts containing exon  <dig> or exon  <dig> are expressed in hek <dig> cells.
exon  <dig> containing transcripts are coordinately and coherently regulated with other bcl2l <dig> transcripts by tsa in hek <dig> cells
the biological significance of the newly identified putative bcl2l <dig> promoter would be further strengthened if transcripts derived from it could be shown to be co-regulated together with other bcl2l <dig> transcripts, in a cellular context where bcl2l <dig> mrna levels are known to be modulated.
in order to investigate this possibility, the levels of different bcl2l <dig> mrnas were quantitatively measured in cdna from hek <dig> cells or hek <dig> cells treated with a known inducer of bcl2l <dig> expression in human cells, namely the histone deacetylase inhibitor trichostatin a   <cit> , using the primer pairs mentioned above.
in hek <dig> cells, exon  <dig> containing bcl2l <dig> transcripts are rapidly and transiently induced by trichostatin a .
in hek <dig> cells, bcl2l <dig> transcripts comprising exon  <dig> sequences  are by far the most abundant .
interestingly, the steady-state levels of human bcl2l <dig> transcripts comprising exons  <dig> and  <dig> are rapidly and transiently increased by tsa, confirming the reported regulation of bcl2l <dig> expression by this inhibitor of histone deacetylases  <cit> .
we next investigated the relative contribution of bcl2l <dig> transcripts comprising exons  <dig> and  <dig> to total bcl2l <dig> transcript levels .
exon 1-containing transcripts display the same induction profile of transcripts detected by a primer pair presumably recognizing most known bcl2l <dig> transcripts .
exon 2-containing transcripts account for the majority of steady-state bcl2l <dig> transcripts in hek <dig> cells.
in untreated hek <dig> cells, the cumulative levels of steady-state bcl2l <dig> transcripts encoding exons 1– <dig> are lower than levels of steady-state bcl2l <dig> transcripts detected using a primer set potentially recognizing most bcl2l <dig> transcripts , and account for approximately 60% of these.
these differences may result from differential stability of bcl2l <dig> transcripts bearing different 5'ends, or from other as yet uncharacterized reasons such as the presence of further promoters in this locus.
collectively, these expression data support the hypothesis that p1-derived transcripts are a bona-fide human bcl2l <dig> mrnas displaying a pattern of regulation which is coherent with the overall regulation of bcl2l <dig> expression by a known small molecule modulator, at least in hek <dig> cells.
e2f regulates the activity of bcl2l11-p1
e2f is a known regulator of bcl2l <dig> transcription during apoptosis  <cit> , as evidenced by studies performed on the rat and mouse bcl2l <dig> loci.
in order to determine if e2f can regulate the expression of human bcl2l <dig> promoters in hek <dig> cells, the real time primers employed above were used to measure levels of bcl2l <dig> transcripts originating from the two known human promoters  in response to transient transfection of a commercially available human e2f expression construct.
this was made possible by the high transfection efficiency achieved in hek <dig> cells with standard lipofection-mediated methodologies .
as indicated in fig.
7a, overexpression of e2f results in a significant increase in the steady-state levels of exon 1-comprising transcripts, suggestive of a role for e2f in regulating the transcription, processing or stability of endogenous p1-derived transcripts.
in contrast, exon 2-containing transcripts were not affected, suggesting that the endogenous p <dig> is not significantly modulated by e2f under these experimental conditions.
as the p <dig> promoter is regulated directly or indirectly by e2f in rodents  <cit> , our results suggest that this regulation may not be conserved in humans, at least under these experimental conditions and in hek <dig> cells.
in order to determine if e2f could directly regulate p <dig> activity, we next investigated the capacity of the e2f expression plasmid to modulate the activity of bcl2l11-p1- <dig> .
we observed an induction of reporter activity with bcl2l11-p1- <dig> which was dependent on the concentration of e2f expression plasmid.
conversely, we did not observe such e2f inducibility with a 5' deletion variant of p <dig> lacking all but  <dig> bp of the genomic region upstream of the transcript initiation site .
these data suggested that an e2f-responsive regulatory element is located within p <dig>  between position - <dig> and - <dig> bp.
we therefore searched for candidate e2f binding sites in the bcl2l11-p1- <dig> sequence using a web-based in silico algorithm, and identified a potential e2f element at position - <dig> .
a number of 5' deletion variants of bcl2l11-p1- <dig> were therefore generated by proofreading pcr using the indicated forward primers , and subsequently cloned into pgl3basic to generate the reporter constructs bcl2l11-p1- <dig>  - <dig>  - <dig>  - <dig>  these constructs were then tested for regulation by overexpressed e2f in hek <dig> cells, as described above.
the results  indicated that significant e2f inducibility was lost upon deletion of a region of only  <dig> bp, located between positions - <dig> and - <dig>  and comprising the candidate e2f binding site located at position - <dig>  direct proof of the interaction between e2f and this candidate e2f binding site was therefore sought by electromobility shift assays .
a double stranded oligonucleotide corresponding to the candidate e2f binding site was labeled and its capacity to bind e2f was studied using nuclear extracts from hek <dig> cells transiently transfected with the human e2f expression construct.
competitions with cold double stranded oligonucleotides comprising either a canonical e2f binding site or a binding site for an irrelevant transcription factor  were used in order to determine the specificity of the observed interaction.
as shown in fig.
<dig>  a strong bandshift  is observed using nuclear extracts from hek <dig> cells transfected with the e2f expression plasmid.
a much weaker but identically migrating band is also visible in nuclear extracts from untransfected hek <dig> cells or from cells transfected with a control plasmid.
competition with excess specific or unspecific competitors indicates that the observed band is specifically competed by an oligonucleotide comprising a canonical e2f binding site .
therefore, the genomic element identified in bcl2l11-p <dig> at position - <dig> as a potential e2f binding site is indeed a functional e2f binding site and provides a molecular basis for e2f regulation of bcl2l11-p <dig>
discussion
we have identified a novel putative promoter and associated exon of the human bcl2l <dig> locus in hek <dig> cells and showed regulation of this promoter by the transcription factor e2f, which is known to modulate bcl2l <dig> transcription in other species.
bcl2l <dig> transcripts derived from this promoter are widely expressed in human tissues, and contribute significantly to bcl2l <dig> expression in testis, heart and liver.
the real-time pcr data presented here constitutes the first systematic, quantitative analysis of steady-state bcl2l <dig> transcript expression levels in different human tissues.
overall, the data obtained on the distribution of bcl2l <dig> mrna levels in different human tissues correlate with published data on the distribution of bcl2l <dig> rna and protein in human tissues, which indicates relatively higher expression in testis and spleen  <cit> , organs where bcl2l <dig> has recognized functions in spermatogenesis and hematopoiesis, respectively  <cit> .
interestingly, the analysis presented in this manuscript indicates that the highest expression levels of bcl2l <dig> transcripts were detected in pancreas, placenta and thyroid tissue, suggesting an important role for bcl2l <dig> in the normal physiology of these tissues.
with respect to p1-derived transcripts, up to 70% of total bcl2l <dig> transcripts are contributed from the newly identified promoter in the testis, an organ where bcl2l <dig> activity was shown to contribute critically to spermatogenesis  <cit> , suggestive for a prominent role for the putative bcl2l <dig> p <dig> in the expression of bcl2l <dig> in this tissue.
although the biological significance of bcl2l <dig> p <dig> remains to be determined, the expression of p1-derived transcripts in various human tissues of relevance to bcl2l <dig> biological function argues for a physiological significance of such transcripts.
the biological significance of the newly identified putative bcl2l <dig> promoter was strengthened by the observation that transcripts derived from it could be shown to be co-regulated together with other bcl2l <dig> transcripts by a known pharmacological modulator of bcl2l <dig> expression , in the same cell line where such transcripts have been identified.
another piece of circumstantial evidence pointing to a physiological role for bcl2l <dig> p <dig> in bcl2l <dig> expression is its regulation by the transcription factor e2f.
studies performed on the rodent bim promoters  have identified this transcriptional regulator as a modulator of bcl2l <dig> expression during apoptotic processes  <cit> .
through a combination of reporter assays and dna-protein interaction studies, we were able to demonstrate that e2f can upregulate bcl2l <dig> p <dig> activity in hek <dig> cells through a cis-acting element identified through a bioinformatics analysis, which can bind e2f in emsa assays and whose presence is required for responsiveness to overexpressed e2f.
this is the first indication of direct transcriptional regulation of a bcl2l <dig> promoter by e2f, as previous reports of e2f involvement in the regulation of bcl2l <dig> expression have shown either an indirect regulation through other transcription factors  <cit>  or have not investigated the underlying mechanism  <cit> .
the potential contribution of p <dig> activity during bcl2l11-mediated apoptotic processes induced by e2f may therefore be physiologically important and deserves further investigation.
a number of further aspects are worthy of further analysis.
first, further characterization of bcl2l <dig> p <dig> in different cellular contexts/backgrounds is required, in particular in contexts where bcl2l <dig> expression is higher than that observed in hek <dig> cells where bcl2l <dig> expression is relatively low, in order to confirm that such new putative promoter is indeed generally active and drives bcl2l <dig> transcripts in most situations where the locus is transcribed.
second, the relative contribution of p1- and p2-derived transcripts to bcl2l <dig> protein expression may differ and needs to be investigated, as e <dig> and e <dig> are alternatively spliced and may contribute differently to mrna stability and translation.
finally, it remains to be determined if a homologue of bcl2l11-p <dig> exists in rodents.
interestingly, a blast analysis using  <dig> kb of mouse genomic sequences upstream of the first coding exon of the murine bcl2l <dig> locus revealed a similar pattern of ests as that evidenced in this manuscript for the human locus, suggesting the potential existence of a bcl2l11-p <dig> homologue in mice  and providing further indirect evidence of an important physiological role for p <dig> in bcl2l <dig> expression.
CONCLUSIONS
through a combined bioinformatics and experimental approach we have identified a novel putative promoter and associated untranslated exon in the human bcl2l <dig> genomic locus, upstream of the previously characterized promoter.
this candidate promoter generates transcripts which are widely expressed in human tissues, and which contribute significantly to overall bcl2l <dig> expression in tissues where bcl2l <dig> is known to play important physiological roles, such as the testis.
our data are suggestive of a complex pattern of transcriptional regulation of the human bcl2l <dig> locus, comprising alternative promoter usage and alternatively spliced 5' untranslated exons, and suggests the presence of additional, as yet unidentified promoter.
although, the general relevance and impact of bcl2l11-p <dig> and of its regulation by e2f in other human cell lines or in vivo remains to be determined, the present study offers novel insights into the structure and regulation of the human bcl2l <dig> locus.

 the human bcl2l locus, located on chromosome 2q encodes a protein of amino acids structurally and functionally related to the bh3-only group of pro-apoptotic bcl family members. the gene is frequently mutated in diverse human tumours leading to loss of bcl2l activity. expression of bcl2l is induced by a diverse range of apoptotic stimuli such as deprivation of growth factors/cytokines, ionizing radiation, and cytotoxic peptides. although the regulation of bcl2l activity is highly complex and is cell context-dependent, studies aimed at identifying the molecular mechanisms underlying bcl2l activation during apoptosis have revealed an important role for the transcriptional regulation of bcl2l gene expression. a genomic region displaying promoter activity has been characterized for the human bcl2l locus, while in the rat the existence of three alternative promoter sequences has been postulated, the most upstream of which corresponds to the promoter described for the human bcl2l gene. regulation of this conserved bcl2l promoter by foxo and e2f has been described using rat bcl2l genomic constructs, thus providing a likely mechanism for the induction of bcl2l expression during programmed cell death, through the involvement of these transcription factors whose activity is induced in apoptotic contexts. whether regulation by e2f and foxo factors is conserved in humans remains to be demonstrated; however, such studies are hampered by the relatively poor inter-specific sequence conservation in non-coding sequences and the paucity of information on the structure and regulation of the human bcl2l locus. we therefore set out to investigate the existence of as yet uncharacterized human bcl2l promoter regions, and to determine the conservation from rodents to man of e2f regulation of bcl2l expression.  identification of a novel putative bcl2l promoter region and associated bcl2l exon in order to further investigate genomic sequences of the human bcl2l locus with promoter activity, we first applied a bioinformatics approach to identify clusters of human ests with comparable 5' ends upstream of the first coding exon of the human bcl2l locus, informative of the possible existence of distinct transcript initiation sites. we analyzed 13kb of human bcl2l genomic sequence immediately upstream of the first bcl2l coding exon by blast against the genbank human est database, and identified clusters with identical/comparable 5'ends. the largest group corresponded well to the human bcl2l promoter already characterized, and conserved between human and rat bcl2l loci. a second, more heterogeneous group localized to a region comprising sequences described in the rat as potentially harbouring alternative promoters. a third group localized to a region ca. kb upstream of the atg, which did not correspond to known bcl2l locus sequences in either man or rodents. this group could be subdivided into two subgroups, each with identical or nearly identical 5' ends, apparently running in opposite directions and potentially suggestive of the existence of a bidirectional promoter. the first indication of the likely existence of an as yet uncharacterized bcl2l promoter associated with novel bcl2l untranslated exon sequences came from the presence, within ests group of one est which clearly comprised the first bcl2l coding exon, but not the first known bcl2l untranslated exon. instead, novel untranslated exon sequences 5' of the first known untranslated bcl2l exon were present, which overlapped with group subgroup b ests. this was suggestive of the presence of a novel bcl2l promoter and associated untranslated exon 5' of the most upstream known bcl2l promoter. we therefore focused on group subgroup b ests, as these might represent novel bcl2l transcribed sequences by virtue of their overlapping with est db which comprises the first bcl2l coding exon. their experimental association with the first bcl2l coding exon in a transcribed sequence would in fact confirm the existence of a new bcl2l promoter and associated untranslated exon. therefore, a series of rt-pcr experiments were performed where the forward primer was anchored within group subgroup b ests and the reverse primer was anchored within the first coding exon of the bcl2l locus, downstream of the translational initiation codon. complementary dna derived from hek mrna was used in these experiments, as these cells were shown to express bcl2l transcripts. the sequence of the resulting amplicons confirmed the presence of bcl2l coding sequences in the amplified cdna. the alignment of the obtained rt-pcr product sequences with db and with representative group subgroup b ests confirmed the co-linearity of these transcribed sequences. this finding confirmed that group subgroup b ests indeed comprise novel untranslated bcl2l sequences, thus identifying a novel bcl2l untranslated exon and confirming the existence of at least one novel putative bcl2l promoter upstream of the previously characterized human bcl2l promoter sequences. having ascertained that group subgroup b ests correspond to bona fide bcl2l transcripts, we next sought to determine the 5' end of such transcripts in order to determine a putative transcript initiation site, defining a possible 5' exon boundary and a starting point for the definition of a promoter region for p 5'race experiments performed on hek cells, using a reverse primer anchored within the novel human bcl2l exon and overlapping with the forward primer used in the rt-pcr studies described above, identified a sequence around the 5' end of group subgroup b ests as the transcript initiation site in hek cells, thus defining the likely 5' boundary of this novel human bcl2l exon and confirming the existence of a novel human bcl2l promoter. a diagram summarizing the position of ests and of the rt-pcr and 5'race products relatively to human bcl2l genomic sequences is provided in fig. 4a. as transcripts originating from this region do not contain sequences associated with the exon immediately downstream of the published human bcl2l promoter, but splice directly onto the first bcl2l coding exon, it follows that the human bcl2l locus might comprise at least two alternative promoters and that alternative splicing occurs in the 5' untranslated exons. a third promoter region may also exist based on data from the rodent bcl2l locus. next, the capacity of p sequences to drive gene expression was tested in a series of reporter assays in hek cells. a human genomic region of bp region comprising a portion of exon was therefore amplified, cloned, sequenced and subcloned into the promoter-less luciferase reporter vector pgl3basic in both orientations, to give rise to the reporter constructs pbcl2l11-p1- and. the constructs were then transiently transfected into hek cells and the resulting luciferase activity compared to basal pgl3basic activity. the results indicate that p sequences can drive gene expression, as measured by luciferase activity, though independently of their orientation. although orientation-dependent activity is usually considered a hallmark of promoter sequences, examples of bona-fide bidirectional promoters are known. therefore, p may represent a promoter with potential for bi-directional activity, at least in hek cells. this possibility is also supported by the existence of group subgroup a ests as discussed above, representing ests with similar 5' ends but with apparently displaying sequences which are complementary to the sense p direction and starting within ca. bp of the transcript initiation site of e although the existence of a bi-directional promoter in this genomic region is supported by our experimental data and from the existence of coherent ests, further studies are required to confirm this possibility. as our interest was on the identification of putative novel bcl2l promoter sequences, this line was not pursued further. exon containing transcripts are widely expressed in human tissues in order to evaluate the possible significance of bcl2l transcripts originating from this novel candidate promoter for overall bcl2l transcript expression in man, the expression of bcl2l mrnas bearing the alternatively spliced exon or exon sequences, or of total bcl2l transcripts, was evaluated in a panel of cdnas derived from human tissues by real-time pcr, using sybr green technology. primer pairs specific for bcl2l exon or or for a bcl2l transcribed region presumably present in most characterized bcl2l transcripts were employed. the results indicated that transcripts originating from the candidate novel bcl2l promoter p were readily detectable in a number of human tissues, and contributed most significantly to bcl2l mrna levels in testis, heart and liver. next, the relative contribution of exon 1- and exon 2-containing transcripts to total bcl2l expression in hek cells was then evaluated by real-time pcr, using sybr green technology. the results again indicated that bcl2l transcripts containing exon or exon are expressed in hek cells. exon containing transcripts are coordinately and coherently regulated with other bcl2l transcripts by tsa in hek cells the biological significance of the newly identified putative bcl2l promoter would be further strengthened if transcripts derived from it could be shown to be co-regulated together with other bcl2l transcripts, in a cellular context where bcl2l mrna levels are known to be modulated. in order to investigate this possibility, the levels of different bcl2l mrnas were quantitatively measured in cdna from hek cells or hek cells treated with a known inducer of bcl2l expression in human cells, namely the histone deacetylase inhibitor trichostatin a, using the primer pairs mentioned above. in hek cells, exon containing bcl2l transcripts are rapidly and transiently induced by trichostatin a. in hek cells, bcl2l transcripts comprising exon sequences are by far the most abundant. interestingly, the steady-state levels of human bcl2l transcripts comprising exons and are rapidly and transiently increased by tsa, confirming the reported regulation of bcl2l expression by this inhibitor of histone deacetylases. we next investigated the relative contribution of bcl2l transcripts comprising exons and to total bcl2l transcript levels. exon 1-containing transcripts display the same induction profile of transcripts detected by a primer pair presumably recognizing most known bcl2l transcripts. exon 2-containing transcripts account for the majority of steady-state bcl2l transcripts in hek cells. in untreated hek cells, the cumulative levels of steady-state bcl2l transcripts encoding exons 1– are lower than levels of steady-state bcl2l transcripts detected using a primer set potentially recognizing most bcl2l transcripts, and account for approximately 60% of these. these differences may result from differential stability of bcl2l transcripts bearing different 5'ends, or from other as yet uncharacterized reasons such as the presence of further promoters in this locus. collectively, these expression data support the hypothesis that p1-derived transcripts are a bona-fide human bcl2l mrnas displaying a pattern of regulation which is coherent with the overall regulation of bcl2l expression by a known small molecule modulator, at least in hek cells. e2f regulates the activity of bcl2l11-p1 e2f is a known regulator of bcl2l transcription during apoptosis, as evidenced by studies performed on the rat and mouse bcl2l loci. in order to determine if e2f can regulate the expression of human bcl2l promoters in hek cells, the real time primers employed above were used to measure levels of bcl2l transcripts originating from the two known human promoters in response to transient transfection of a commercially available human e2f expression construct. this was made possible by the high transfection efficiency achieved in hek cells with standard lipofection-mediated methodologies. as indicated in fig. 7a, overexpression of e2f results in a significant increase in the steady-state levels of exon 1-comprising transcripts, suggestive of a role for e2f in regulating the transcription, processing or stability of endogenous p1-derived transcripts. in contrast, exon 2-containing transcripts were not affected, suggesting that the endogenous p is not significantly modulated by e2f under these experimental conditions. as the p promoter is regulated directly or indirectly by e2f in rodents, our results suggest that this regulation may not be conserved in humans, at least under these experimental conditions and in hek cells. in order to determine if e2f could directly regulate p activity, we next investigated the capacity of the e2f expression plasmid to modulate the activity of bcl2l11-p1-. we observed an induction of reporter activity with bcl2l11-p1- which was dependent on the concentration of e2f expression plasmid. conversely, we did not observe such e2f inducibility with a 5' deletion variant of p lacking all but bp of the genomic region upstream of the transcript initiation site. these data suggested that an e2f-responsive regulatory element is located within p between position - and - bp. we therefore searched for candidate e2f binding sites in the bcl2l11-p1- sequence using a web-based in silico algorithm, and identified a potential e2f element at position -. a number of 5' deletion variants of bcl2l11-p1- were therefore generated by proofreading pcr using the indicated forward primers, and subsequently cloned into pgl3basic to generate the reporter constructs bcl2l11-p1- - - - these constructs were then tested for regulation by overexpressed e2f in hek cells, as described above. the results indicated that significant e2f inducibility was lost upon deletion of a region of only bp, located between positions - and - and comprising the candidate e2f binding site located at position - direct proof of the interaction between e2f and this candidate e2f binding site was therefore sought by electromobility shift assays. a double stranded oligonucleotide corresponding to the candidate e2f binding site was labeled and its capacity to bind e2f was studied using nuclear extracts from hek cells transiently transfected with the human e2f expression construct. competitions with cold double stranded oligonucleotides comprising either a canonical e2f binding site or a binding site for an irrelevant transcription factor were used in order to determine the specificity of the observed interaction. as shown in fig. a strong bandshift is observed using nuclear extracts from hek cells transfected with the e2f expression plasmid. a much weaker but identically migrating band is also visible in nuclear extracts from untransfected hek cells or from cells transfected with a control plasmid. competition with excess specific or unspecific competitors indicates that the observed band is specifically competed by an oligonucleotide comprising a canonical e2f binding site. therefore, the genomic element identified in bcl2l11-p at position - as a potential e2f binding site is indeed a functional e2f binding site and provides a molecular basis for e2f regulation of bcl2l11-p discussion we have identified a novel putative promoter and associated exon of the human bcl2l locus in hek cells and showed regulation of this promoter by the transcription factor e2f, which is known to modulate bcl2l transcription in other species. bcl2l transcripts derived from this promoter are widely expressed in human tissues, and contribute significantly to bcl2l expression in testis, heart and liver. the real-time pcr data presented here constitutes the first systematic, quantitative analysis of steady-state bcl2l transcript expression levels in different human tissues. overall, the data obtained on the distribution of bcl2l mrna levels in different human tissues correlate with published data on the distribution of bcl2l rna and protein in human tissues, which indicates relatively higher expression in testis and spleen, organs where bcl2l has recognized functions in spermatogenesis and hematopoiesis, respectively. interestingly, the analysis presented in this manuscript indicates that the highest expression levels of bcl2l transcripts were detected in pancreas, placenta and thyroid tissue, suggesting an important role for bcl2l in the normal physiology of these tissues. with respect to p1-derived transcripts, up to 70% of total bcl2l transcripts are contributed from the newly identified promoter in the testis, an organ where bcl2l activity was shown to contribute critically to spermatogenesis, suggestive for a prominent role for the putative bcl2l p in the expression of bcl2l in this tissue. although the biological significance of bcl2l p remains to be determined, the expression of p1-derived transcripts in various human tissues of relevance to bcl2l biological function argues for a physiological significance of such transcripts. the biological significance of the newly identified putative bcl2l promoter was strengthened by the observation that transcripts derived from it could be shown to be co-regulated together with other bcl2l transcripts by a known pharmacological modulator of bcl2l expression, in the same cell line where such transcripts have been identified. another piece of circumstantial evidence pointing to a physiological role for bcl2l p in bcl2l expression is its regulation by the transcription factor e2f. studies performed on the rodent bim promoters have identified this transcriptional regulator as a modulator of bcl2l expression during apoptotic processes. through a combination of reporter assays and dna-protein interaction studies, we were able to demonstrate that e2f can upregulate bcl2l p activity in hek cells through a cis-acting element identified through a bioinformatics analysis, which can bind e2f in emsa assays and whose presence is required for responsiveness to overexpressed e2f. this is the first indication of direct transcriptional regulation of a bcl2l promoter by e2f, as previous reports of e2f involvement in the regulation of bcl2l expression have shown either an indirect regulation through other transcription factors or have not investigated the underlying mechanism. the potential contribution of p activity during bcl2l11-mediated apoptotic processes induced by e2f may therefore be physiologically important and deserves further investigation. a number of further aspects are worthy of further analysis. first, further characterization of bcl2l p in different cellular contexts/backgrounds is required, in particular in contexts where bcl2l expression is higher than that observed in hek cells where bcl2l expression is relatively low, in order to confirm that such new putative promoter is indeed generally active and drives bcl2l transcripts in most situations where the locus is transcribed. second, the relative contribution of p1- and p2-derived transcripts to bcl2l protein expression may differ and needs to be investigated, as e and e are alternatively spliced and may contribute differently to mrna stability and translation. finally, it remains to be determined if a homologue of bcl2l11-p exists in rodents. interestingly, a blast analysis using kb of mouse genomic sequences upstream of the first coding exon of the murine bcl2l locus revealed a similar pattern of ests as that evidenced in this manuscript for the human locus, suggesting the potential existence of a bcl2l11-p homologue in mice and providing further indirect evidence of an important physiological role for p in bcl2l expression.  through a combined bioinformatics and experimental approach we have identified a novel putative promoter and associated untranslated exon in the human bcl2l genomic locus, upstream of the previously characterized promoter. this candidate promoter generates transcripts which are widely expressed in human tissues, and which contribute significantly to overall bcl2l expression in tissues where bcl2l is known to play important physiological roles, such as the testis. our data are suggestive of a complex pattern of transcriptional regulation of the human bcl2l locus, comprising alternative promoter usage and alternatively spliced 5' untranslated exons, and suggests the presence of additional, as yet unidentified promoter. although, the general relevance and impact of bcl2l11-p and of its regulation by e2f in other human cell lines or in vivo remains to be determined, the present study offers novel insights into the structure and regulation of the human bcl2l locus. 
in the search for novel promoter sequences in the human bcl2l locus, we have identified previously unrecognized genomic sequences displaying promoter activity and e2f responsiveness, and driving the expression of bcl2l coding transcripts. in hek cells, this novel candidate promoter originates bcl2l transcripts whose expression can be modulated by a known modulator of bcl2l expression and by e2f, a characterized transcriptional regulator of bcl2l expression.
 despite the importance of the bcl2l protein in various apoptotic processes in development and disease, little is known of the promoter structure of the human bcl2l locus and of the cis-acting elements regulating expression of the human gene.  in the search for novel promoter sequences in the human bcl2l locus, we have identified previously unrecognized genomic sequences displaying promoter activity and e2f responsiveness, and driving the expression of bcl2l coding transcripts. in man, transcripts originating from this novel putative promoter contribute significantly to total bcl2l mrna expression in testis, heart and liver. in hek cells, this novel candidate promoter originates bcl2l transcripts whose expression can be modulated by a known modulator of bcl2l expression and by e2f, a characterized transcriptional regulator of bcl2l expression.  the identification of a novel putative human bcl2l promoter provides new insights into the structure and regulation of the bcl2l locus. 