BACKGROUND
the metazoan genome is composed of coding sequences flanked by non-coding regions containing regulatory elements.
these elements consist of short nucleotide sequences, which program gene expression at a given time and in a specific cell  <cit> .
despite the significant progress in genomics, which has led to the identification of most genes of the human genome, the knowledge of transcriptional regulation remains unclear.
our promoter study depends on a set of bioinformatics tools and allows the study of mechanisms of this genome regulation  <cit> .
it relies on an analysis of gene promoter sequences which would include a combination  of several transcription factor binding sites  ranging in length from ten to fifty nucleotides.
tfbs are recognized by transcription factors , which allow the activation or repression of gene transcription.
tfbs are difficult to differentiate from non-functional random genomic sequences  <cit> .
these regulatory sequences and the genes that they govern define the functional spaces in the genome.
thus, such elements play a major role in the development, the environmental adaptation, the response to nutritional uptake and the pathogenesis.
the adenine nucleotide translocator , also referred to by the generic term "adp/atp carrier" , is a protein encoded from the nuclear genome and inserted in the inner mitochondrial membrane.
ant allows the exchange of atp and adp adenylic nucleotides between the mitochondrial matrix and the cytoplasm.
such a function is of primary importance because the ant would be the main protein of the inner mitochondrial membrane able to convey this energy.
the importance of this ant protein is stressed by the fact that there exists, from yeasts to humans, four isoforms with similar amino-acid sequences , with different kinetic properties, encoded from four independent genes, each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proli-feration.
this allows the energy production to adapt to the metabolic parameters linked to the cellular environment and cell cycle  <cit> .
the peptide sequences of these four isoforms are very close ; they differ only by several amino acids which are involved in the atp and adp interaction sites.
the specific transcriptional regulation of each of the four ant genes is an interesting example of multi-isoform gene regulation.
the metabolic and physiological consequences of these molecular regulatory mechanisms play a major role in the evolution of cellular metabolic pathways.
each of the four isoforms is known to play a specific role in cellular bioenergetics: ant <dig>  provides mitochondrial atp for heart and skeletal muscle contraction  <cit> .
the kinetic properties of this ant <dig> isoform allow the rapid and massive mitochondrial atp export required for muscular contraction.
the second isoform, ant <dig> , is weakly or not at all expressed in human tissues and maintains the intra-mitochondrial functions under glycolytic conditions required in proliferative cells  <cit> .
ant <dig> is known to have a function opposite to that of ant <dig> by transporting glycolytic atp toward the mitochondrial matrix  <cit> .
we identified a specific regulatory sequence in the promoter region of the human ant <dig> gene: the grbox element  upstream of the tss   <cit> .
ant <dig>  is the constitutively expressed ubiquitous isoform that is integrated into the mitochondrial membrane when no other isoform is produced  <cit> .
in rodents, the ant <dig> gene was lost during evolution.
it is possible that, unlike humans, rodent physiology does not require two isoforms with different kinetics .
this assumption would be supported by the disappearance, in the rodents, of the oxbox regulatory element from the ant <dig> promoter  <cit> , which would determine the muscle specific expression of this isoform.
the last isoform, ant <dig> , was recently identified in humans, and is expressed mainly in the testicle  <cit> .
this isoform appears in mammals and is essential during spermatogenesis  <cit> .
its peptide sequence is very similar  to that of the other ant isoforms.
the main characteristic of the ant <dig> isoform is the presence of additional peptides, specifically the n-  and c-  terminal sequences, which the other three isoforms lack.
the proposed hypothesis for the role of this isoform is that it compensates for the loss of the ant <dig> gene function  during male meiosis  <cit> .
a recent computational analysis enabled us to propose a specific role for the ant <dig> isoform in spermatozoid bioenergetics  <cit> .
we pursued and developed this computational analysis to compare the mechanisms of transcriptional regulation of the four ant isoforms through analysis of nucleotide sequences upstream of the supposed tss.
the nucleotide sequences of these promoter regions from several mammalian species were compared to follow the phylogeny of specific sequences of transcriptional regulation.
promoter sequences preserved throughout evolution might be of major importance to the survival of the organism  <cit> .
this study is based on a combination of software and databases including those available on-line, such as genomatix  <cit>  and ensembl  <cit> , geneprom from our laboratory.
this analysis led to interesting conclusions linking promoter structure and co-regulation of a set of genes.
RESULTS
an outline of the bioinformatics pipeline implemented for ant sequences analysis is illustrated in figure  <dig>
alignment and selection of ant promoter sequences from various mammalian species
mammalian species were screened for the homologous ant gene sequences.
thirty mammalian species were screened for the four ant genes and all ant sequences  were imported.
the selected species corresponded to the  <dig> available mammalian sequences when the sequences were searched on ensembl  <cit> .
the mammalian sequences from each ant gene were aligned using the clustal software and several sequences were selected according to the correct overlap of their 5’ upstream first exon sequences.
mammalian sequences with gaps, incomplete sequences or with more than 10% unknown nucleotides were discarded.
eight sequences  were obtained for the ant <dig> gene,  <dig> for the ant <dig> gene,  <dig> for the ant <dig> gene and  <dig> for the ant <dig> gene.
the evolutionary history was inferred using the neighbor-joining method  <cit>  with the bootstrap test  <cit>  and with the maximum composite likelihood method for evolutionary distances  <cit>  .
evolutionary analyses were conducted in mega <dig>  <cit> .
identification of transcriptional matrices shared by the ant <dig> to  <dig> gene sequences
using the frameworker tool from genomatix, we found several matrices shared by the selected mammalian promoter sequences of each ant gene.
human models were constructed  using different sets of parameters .
an example of model is shown in the promoter sequence of each isoform .
the distance between the matrices and nucleotide strings was set to twice the distance given for the corresponding human ant promoter by default.
matrix families  or matrices  with an asterisk were identified from phylogenetic analyses and showed to be conserved in orthologous promoters .
the other matrices are selected with the genomatix matinspector tool on the human promoter sequences.
most matrices and iupac strings from the ant <dig> models are directly or indirectly involved in muscle cell growth and differentiation.
most of those from the ant <dig> models found were involved in cell growth and proliferation .
based on phylogenetic analysis, transcriptional factors involved in ant <dig> gene regulation appear to serve different major functions.
however, contrary to the three other ant genes , the stringency of the ant <dig> models required to identify co-regulated genes was too low to lead to conclusive results .
lastly, most matrices and iupac strings from the ant <dig> models are involved in testis development, spermatogenesis or the glycolytic energetic metabolism.
screening of genes co-regulated with each ant
constructed models of each ant gene promoter were screened as described in figure  <dig>  either on the whole chromosome human sequences or the human promoter database  using genomatix modelinspector and geneprom.
only genes with a model located next to their transcription start sites were selected .
a match for all matrices and iupac strings and in the same order is required.
moreover, several locations  in clones without identified genes were not retained.
encoded protein data were obtained from the ensembl database  <cit>  and data on protein function were obtained from uniprot.
<cit>  .
results with ant <dig> models : twelve genes revealed from screening with the ant <dig> promoter models  were identified.
all genes are specifically expressed in muscle or directly involved in bioenergetic metabolism.
seven of these genes were found to be overexpressed in microarrays data.
several constructed models of the ant <dig> promoter region were screened as described in figure  <dig> either on the full chromosomal human sequences or the human promoter library .
genes in bold are shown overexpressed in microarrays .
gene ids are with  <dig> numbers .
results with ant <dig> models : seventeen genes were identified by modelinspector / geneprom analyses.
ten of them are expressed in conditions directly related to cell proliferation and glycolytic metabolism and five have a proposed function which appears to not be directly linked to one another.
twelve genes were found to be overexpressed in microarrays data .
several constructed models of the ant <dig> promoter region were screened as described in figure  <dig> either on the whole chromosome human sequences or the human promoter library .
function involved in cell proliferation is shown in bold characters.
genes in bold are shown overexpressed in microarrays .
δno correct probe set on affymetrix chips corresponding to this gene.
gene ids are with  <dig> numbers .
results with ant <dig> models : no candidate genes were identified with regular stringency .
a lower stringency led to identify  <dig> genes with the modelinspector and geneprom analyses.
five have unknown function and the remaining  <dig> have heterogeneous functions not linked to bioenergetic pathways.
the sole gene found overexpressed in testes microarrays, ift <dig>  encodes for an intraflagellar transport protein involved in cilium biogenesis.
this gene appears as a false positive result.
results with ant <dig> models :  <dig> genes were identified by modelinspector/geneprom analyses.
four of these genes have an unknown or a supposed function;  <dig> are specifically expressed in the testis, during spermatogenesis, or involved in prostate metabolism.
thirteen genes were found to be overexpressed in microarrays data .
several constructed models of the ant <dig> promoter region were screened as described in figure  <dig> either on the full chromosomal human sequences or the human promoter library .
function involved in spermatogenesis or in testis or prostate metabolism is shown in bold characters.
genes in bold are shown overexpressed in microarrays .
§genes identified in the previous modelinspector / geneprom analysis  <cit> .
δno correct probe set on affymetrix chips corresponding to this gene.
if numerical expression results were compared for the  <dig> ant isoform genes , the expression levels were similar in glioblastoma versus control tissue for the ubiquitous ant <dig> isoform gene and the identified co-regulated genes.
in the opposite, the regulated ant <dig>   <dig> and  <dig> genes were over-expressed in the corresponding tissues
discussion
we built a pipeline of bioinformatics analyses for the study of the transcriptional regulation of a set of genes and the prediction of co-regulated genes.
co-regulated genes could encode proteins involved in the same metabolic network including an entire set of different pathways.
this analysis was designed for, in the longer term, predicting a precise signature of a cellular metabolic change, which is the consequence of physiological conditions, disease, or a response to a specific pharmacological or nutritional treatment.
this pipeline can analyse the structure of the promoter region of a gene and the construction of regulatory models composed of a combination of several small nucleotide sequences specifically linked to the gene function.
the strategy of the promotology analysis that we adopted relies on the crossing of three complementary analyses:  <dig> – screening of genes located next to the constructed models on the whole human genome: combination of the genomatix modelinspector tools and geneprom software)  <cit> ;  <dig> – screening of these models on a database of human promoter sequences by modelinspector / human promoters;  <dig> - screening of selected models in several mammalian species using the “search for phylogenetically conserved promoter models” tool).
the crossing of these three analyses allowed us to identify, with higher stringency, a limited set of genes controlled by the same model of promoter sequence.
this bioinformatics protocol was tested on a set of genes encoding four isoforms of the ant protein , each having a specific role in a specific cellular type.
three of these four proteins  are controlled at the transcriptional level by a specific mechanism.
the fourth  is the ubiquitous isoform constitutively expressed in all cells.
the implementation of our promotology analysis on this set of four ant genes accounted for a powerful validation of our strategy:
ant1: the gene encoding the isoform specifically expressed in muscle tissues  <cit>  enabled us to build five models of the promoter sequence .
these models are found in the promoters of  <dig> genes highly expressed in muscle tissues and / or involved in bioenergetic metabolism and having a direct connection with muscle cell metabolism or mitochondrial atp synthesis .
in particular,  <dig> of these genes encode proteins that are included to  <dig> of the mitochondrial complexes of oxidative phosphorylation: nadh dehydrogenase , cytochrome oxidase  and atp synthase   <cit>  .
moreover, another gene carrying a model, coq <dig>  is involved in the synthesis of these complexes  <cit> .
other identified genes encode proteins involved in major pathways of muscle metabolism such as ano1/tmem16a in calcium transport  <cit>  and myo1f in muscle contraction  <cit> .
ant2: most of the  <dig> genes carrying a model resulting from the ant <dig> gene encode proteins that play a role in pathways related to cell division and proliferation  .
several other identified genes encode signalling proteins such as cdknÀip, gadd45b or hif1-alpha.
the hif1-alpha-protein is known to induce the transcription of the hkii gene under conditions of glycolytic metabolism  <cit> .
similar to ant <dig>  hkii is involved in the uptake of glycolytic atp through the inner mitochondrial membrane.
this hkii / ant <dig> indirect co-regulation is consistent with their complementary roles in glycolytic conditions   <cit> .
ant3: no gene carrying models built from the ant <dig> gene promoter could be identified if stringency parameters similar to that used for three other isoforms were selected.
analyses with lower stringencies  reveal up to  <dig> genes.
five of these genes encode proteins with an unknown function; the five others have no apparent functional link among one another .
moreover, the analysis of the ant <dig> promoter region by the genomatix matinspector and promoterinspector tools proposed few regulation matrices as compared to genes of the three other regulated isoforms .
in low stringency conditions, the sequence of the model becomes not significant: for a model with  <dig> matrices, the  <dig> to  <dig> nucleotides of the core sequences are conserved with an identity of 100%.
thus, such models could often lead to the identification of unrelated false positive genes.
moreover, this study confirms that our model construction from conserved models in different mammalian species could clearly identify the not specifically regulated ubiquitous genes.
ant4: the models constructed from the ant <dig> gene promoter allowed us to identify  <dig> genes with known function in spermatogenesis .
our previous work on the promoter of this ant <dig> gene led us to identify a part of these co-regulated genes  <cit> .
the new version of our geneprom software enabled us to identify  <dig> new genes also directly related to spermatogenesis.
among these  <dig> genes specifically expressed in the testes, two encode the glucose-6-phosphatases  <dig> and  <dig> enzymes, producing glucose from glucose-6-phosphate, leading to atp synthesis  <cit> .
this function is in complete agreement with the exclusively glycolytic metabolism of spermatozoids  <cit> .
moreover, this glycolytic atp production is also consistent with the expression and the specific role of the ant <dig> isoform in spermatozoid bioenergetics: part of the atp produced by glucose-6-phosphatases  <dig> and  <dig> could be imported into mitochondria by ant <dig>  to compensate for the absence of the ant <dig> isoform    <cit> .
thus, this in silico analysis leads to very interesting conclusions on the relationship between transcriptional regulatory pathways and protein function in a cellular metabolic network.
a set of genes encoding protein isoforms expressed with tissue specificity turned out to be a clear validation of our strategy.
the phylogenetic comparison of promoters allowed for the identification of nucleotidic matrices with major roles in gene regulation.
however, the exclusive use of regulatory matrices has limits; it requires multiple analyses with different parameters of stringency and layout of each matrix compared with the other involved regulatory elements.
the presence, in a promoter region, of described and validated nucleotide sequences, such as the oxbox   <cit>  and grbox   <cit> , known to take part to the regulation of the ant <dig> and ant <dig> genes, respectively, is a crucial argument in the construction of a powerful regulatory model.
the presence of a gene encoding a ubiquitous isoform, ant <dig>  and the failure to identify co-regulated genes with models built from its promoter region allow us to validate our strategy.
moreover, this analysis of an unregulated ubiquitous gene becomes a standard for further works by providing a precise scale of similarity of matrices in our models.
thus, a strategy of promotology, based simultaneously on a conclusive phylogenetic analysis and on already validated regulatory nucleotide sequences, allows for the identification of co-regulated genes.
our work on this set of isoforms also showed that transcriptional regulation is a major mechanism of cellular specificity.
the structure of the promoter sequence directly upstream of the transcription start site itself allows the identification of co-regulated genes.
this suggests that, at least for the regulation of bioenergetic pathways described in this work, other supposed regulatory mechanisms including microrna or the messenger rna stability, would intervene in other cellular functions.
moreover, our strategy allows overcoming the insufficiencies of other techniques used for the study of gene expression.
taking into account the very close coding sequences of the four ant isoforms, no currently commercial microarrays able to simultaneously and specifically quantify the four transcripts is available.
in addition, the very hydrophobic properties of these proteins do not allow their identification by 2d electrophoresis and no specific antibody of each isoform is available.
CONCLUSIONS
in conclusion, our computational strategy on a set of four isoforms, known for their specific functions in cell bioenergetics, enabled us to develop a powerful analysis of gene promoter sequences.
our analyses enabled us to identify an entire set of co-regulated genes, involved in the same cellular function.
this study validates the major role of the proximal promoter region in tissue specificity and should provide, along with transcriptomics and metabolomics, assistance in developing cellular metabolic networks and the study of their regulatory pathways.

 the metazoan genome is composed of coding sequences flanked by non-coding regions containing regulatory elements. these elements consist of short nucleotide sequences, which program gene expression at a given time and in a specific cell. despite the significant progress in genomics, which has led to the identification of most genes of the human genome, the knowledge of transcriptional regulation remains unclear. our promoter study depends on a set of bioinformatics tools and allows the study of mechanisms of this genome regulation. it relies on an analysis of gene promoter sequences which would include a combination of several transcription factor binding sites ranging in length from ten to fifty nucleotides. tfbs are recognized by transcription factors, which allow the activation or repression of gene transcription. tfbs are difficult to differentiate from non-functional random genomic sequences. these regulatory sequences and the genes that they govern define the functional spaces in the genome. thus, such elements play a major role in the development, the environmental adaptation, the response to nutritional uptake and the pathogenesis. the adenine nucleotide translocator, also referred to by the generic term "adp/atp carrier", is a protein encoded from the nuclear genome and inserted in the inner mitochondrial membrane. ant allows the exchange of atp and adp adenylic nucleotides between the mitochondrial matrix and the cytoplasm. such a function is of primary importance because the ant would be the main protein of the inner mitochondrial membrane able to convey this energy. the importance of this ant protein is stressed by the fact that there exists, from yeasts to humans, four isoforms with similar amino-acid sequences, with different kinetic properties, encoded from four independent genes, each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proli-feration. this allows the energy production to adapt to the metabolic parameters linked to the cellular environment and cell cycle. the peptide sequences of these four isoforms are very close; they differ only by several amino acids which are involved in the atp and adp interaction sites. the specific transcriptional regulation of each of the four ant genes is an interesting example of multi-isoform gene regulation. the metabolic and physiological consequences of these molecular regulatory mechanisms play a major role in the evolution of cellular metabolic pathways. each of the four isoforms is known to play a specific role in cellular bioenergetics: ant provides mitochondrial atp for heart and skeletal muscle contraction. the kinetic properties of this ant isoform allow the rapid and massive mitochondrial atp export required for muscular contraction. the second isoform, ant, is weakly or not at all expressed in human tissues and maintains the intra-mitochondrial functions under glycolytic conditions required in proliferative cells. ant is known to have a function opposite to that of ant by transporting glycolytic atp toward the mitochondrial matrix. we identified a specific regulatory sequence in the promoter region of the human ant gene: the grbox element upstream of the tss. ant is the constitutively expressed ubiquitous isoform that is integrated into the mitochondrial membrane when no other isoform is produced. in rodents, the ant gene was lost during evolution. it is possible that, unlike humans, rodent physiology does not require two isoforms with different kinetics. this assumption would be supported by the disappearance, in the rodents, of the oxbox regulatory element from the ant promoter, which would determine the muscle specific expression of this isoform. the last isoform, ant, was recently identified in humans, and is expressed mainly in the testicle. this isoform appears in mammals and is essential during spermatogenesis. its peptide sequence is very similar to that of the other ant isoforms. the main characteristic of the ant isoform is the presence of additional peptides, specifically the n- and c- terminal sequences, which the other three isoforms lack. the proposed hypothesis for the role of this isoform is that it compensates for the loss of the ant gene function during male meiosis. a recent computational analysis enabled us to propose a specific role for the ant isoform in spermatozoid bioenergetics. we pursued and developed this computational analysis to compare the mechanisms of transcriptional regulation of the four ant isoforms through analysis of nucleotide sequences upstream of the supposed tss. the nucleotide sequences of these promoter regions from several mammalian species were compared to follow the phylogeny of specific sequences of transcriptional regulation. promoter sequences preserved throughout evolution might be of major importance to the survival of the organism. this study is based on a combination of software and databases including those available on-line, such as genomatix and ensembl, geneprom from our laboratory. this analysis led to interesting conclusions linking promoter structure and co-regulation of a set of genes.  an outline of the bioinformatics pipeline implemented for ant sequences analysis is illustrated in figure alignment and selection of ant promoter sequences from various mammalian species mammalian species were screened for the homologous ant gene sequences. thirty mammalian species were screened for the four ant genes and all ant sequences were imported. the selected species corresponded to the available mammalian sequences when the sequences were searched on ensembl. the mammalian sequences from each ant gene were aligned using the clustal software and several sequences were selected according to the correct overlap of their 5’ upstream first exon sequences. mammalian sequences with gaps, incomplete sequences or with more than 10% unknown nucleotides were discarded. eight sequences were obtained for the ant gene, for the ant gene, for the ant gene and for the ant gene. the evolutionary history was inferred using the neighbor-joining method with the bootstrap test and with the maximum composite likelihood method for evolutionary distances. evolutionary analyses were conducted in mega. identification of transcriptional matrices shared by the ant to gene sequences using the frameworker tool from genomatix, we found several matrices shared by the selected mammalian promoter sequences of each ant gene. human models were constructed using different sets of parameters. an example of model is shown in the promoter sequence of each isoform. the distance between the matrices and nucleotide strings was set to twice the distance given for the corresponding human ant promoter by default. matrix families or matrices with an asterisk were identified from phylogenetic analyses and showed to be conserved in orthologous promoters. the other matrices are selected with the genomatix matinspector tool on the human promoter sequences. most matrices and iupac strings from the ant models are directly or indirectly involved in muscle cell growth and differentiation. most of those from the ant models found were involved in cell growth and proliferation. based on phylogenetic analysis, transcriptional factors involved in ant gene regulation appear to serve different major functions. however, contrary to the three other ant genes, the stringency of the ant models required to identify co-regulated genes was too low to lead to conclusive results. lastly, most matrices and iupac strings from the ant models are involved in testis development, spermatogenesis or the glycolytic energetic metabolism. screening of genes co-regulated with each ant constructed models of each ant gene promoter were screened as described in figure either on the whole chromosome human sequences or the human promoter database using genomatix modelinspector and geneprom. only genes with a model located next to their transcription start sites were selected. a match for all matrices and iupac strings and in the same order is required. moreover, several locations in clones without identified genes were not retained. encoded protein data were obtained from the ensembl database and data on protein function were obtained from uniprot.. results with ant models: twelve genes revealed from screening with the ant promoter models were identified. all genes are specifically expressed in muscle or directly involved in bioenergetic metabolism. seven of these genes were found to be overexpressed in microarrays data. several constructed models of the ant promoter region were screened as described in figure either on the full chromosomal human sequences or the human promoter library. genes in bold are shown overexpressed in microarrays. gene ids are with numbers. results with ant models: seventeen genes were identified by modelinspector / geneprom analyses. ten of them are expressed in conditions directly related to cell proliferation and glycolytic metabolism and five have a proposed function which appears to not be directly linked to one another. twelve genes were found to be overexpressed in microarrays data. several constructed models of the ant promoter region were screened as described in figure either on the whole chromosome human sequences or the human promoter library. function involved in cell proliferation is shown in bold characters. genes in bold are shown overexpressed in microarrays. δno correct probe set on affymetrix chips corresponding to this gene. gene ids are with numbers. results with ant models: no candidate genes were identified with regular stringency. a lower stringency led to identify genes with the modelinspector and geneprom analyses. five have unknown function and the remaining have heterogeneous functions not linked to bioenergetic pathways. the sole gene found overexpressed in testes microarrays, ift encodes for an intraflagellar transport protein involved in cilium biogenesis. this gene appears as a false positive result. results with ant models: genes were identified by modelinspector/geneprom analyses. four of these genes have an unknown or a supposed function; are specifically expressed in the testis, during spermatogenesis, or involved in prostate metabolism. thirteen genes were found to be overexpressed in microarrays data. several constructed models of the ant promoter region were screened as described in figure either on the full chromosomal human sequences or the human promoter library. function involved in spermatogenesis or in testis or prostate metabolism is shown in bold characters. genes in bold are shown overexpressed in microarrays. §genes identified in the previous modelinspector / geneprom analysis. δno correct probe set on affymetrix chips corresponding to this gene. if numerical expression results were compared for the ant isoform genes, the expression levels were similar in glioblastoma versus control tissue for the ubiquitous ant isoform gene and the identified co-regulated genes. in the opposite, the regulated ant and genes were over-expressed in the corresponding tissues discussion we built a pipeline of bioinformatics analyses for the study of the transcriptional regulation of a set of genes and the prediction of co-regulated genes. co-regulated genes could encode proteins involved in the same metabolic network including an entire set of different pathways. this analysis was designed for, in the longer term, predicting a precise signature of a cellular metabolic change, which is the consequence of physiological conditions, disease, or a response to a specific pharmacological or nutritional treatment. this pipeline can analyse the structure of the promoter region of a gene and the construction of regulatory models composed of a combination of several small nucleotide sequences specifically linked to the gene function. the strategy of the promotology analysis that we adopted relies on the crossing of three complementary analyses: – screening of genes located next to the constructed models on the whole human genome: combination of the genomatix modelinspector tools and geneprom software); – screening of these models on a database of human promoter sequences by modelinspector / human promoters; - screening of selected models in several mammalian species using the “search for phylogenetically conserved promoter models” tool). the crossing of these three analyses allowed us to identify, with higher stringency, a limited set of genes controlled by the same model of promoter sequence. this bioinformatics protocol was tested on a set of genes encoding four isoforms of the ant protein, each having a specific role in a specific cellular type. three of these four proteins are controlled at the transcriptional level by a specific mechanism. the fourth is the ubiquitous isoform constitutively expressed in all cells. the implementation of our promotology analysis on this set of four ant genes accounted for a powerful validation of our strategy: ant1: the gene encoding the isoform specifically expressed in muscle tissues enabled us to build five models of the promoter sequence. these models are found in the promoters of genes highly expressed in muscle tissues and / or involved in bioenergetic metabolism and having a direct connection with muscle cell metabolism or mitochondrial atp synthesis. in particular, of these genes encode proteins that are included to of the mitochondrial complexes of oxidative phosphorylation: nadh dehydrogenase, cytochrome oxidase and atp synthase. moreover, another gene carrying a model, coq is involved in the synthesis of these complexes. other identified genes encode proteins involved in major pathways of muscle metabolism such as ano1/tmem16a in calcium transport and myo1f in muscle contraction. ant2: most of the genes carrying a model resulting from the ant gene encode proteins that play a role in pathways related to cell division and proliferation. several other identified genes encode signalling proteins such as cdknÀip, gadd45b or hif1-alpha. the hif1-alpha-protein is known to induce the transcription of the hkii gene under conditions of glycolytic metabolism. similar to ant hkii is involved in the uptake of glycolytic atp through the inner mitochondrial membrane. this hkii / ant indirect co-regulation is consistent with their complementary roles in glycolytic conditions. ant3: no gene carrying models built from the ant gene promoter could be identified if stringency parameters similar to that used for three other isoforms were selected. analyses with lower stringencies reveal up to genes. five of these genes encode proteins with an unknown function; the five others have no apparent functional link among one another. moreover, the analysis of the ant promoter region by the genomatix matinspector and promoterinspector tools proposed few regulation matrices as compared to genes of the three other regulated isoforms. in low stringency conditions, the sequence of the model becomes not significant: for a model with matrices, the to nucleotides of the core sequences are conserved with an identity of 100%. thus, such models could often lead to the identification of unrelated false positive genes. moreover, this study confirms that our model construction from conserved models in different mammalian species could clearly identify the not specifically regulated ubiquitous genes. ant4: the models constructed from the ant gene promoter allowed us to identify genes with known function in spermatogenesis. our previous work on the promoter of this ant gene led us to identify a part of these co-regulated genes. the new version of our geneprom software enabled us to identify new genes also directly related to spermatogenesis. among these genes specifically expressed in the testes, two encode the glucose-6-phosphatases and enzymes, producing glucose from glucose-6-phosphate, leading to atp synthesis. this function is in complete agreement with the exclusively glycolytic metabolism of spermatozoids. moreover, this glycolytic atp production is also consistent with the expression and the specific role of the ant isoform in spermatozoid bioenergetics: part of the atp produced by glucose-6-phosphatases and could be imported into mitochondria by ant to compensate for the absence of the ant isoform. thus, this in silico analysis leads to very interesting conclusions on the relationship between transcriptional regulatory pathways and protein function in a cellular metabolic network. a set of genes encoding protein isoforms expressed with tissue specificity turned out to be a clear validation of our strategy. the phylogenetic comparison of promoters allowed for the identification of nucleotidic matrices with major roles in gene regulation. however, the exclusive use of regulatory matrices has limits; it requires multiple analyses with different parameters of stringency and layout of each matrix compared with the other involved regulatory elements. the presence, in a promoter region, of described and validated nucleotide sequences, such as the oxbox and grbox, known to take part to the regulation of the ant and ant genes, respectively, is a crucial argument in the construction of a powerful regulatory model. the presence of a gene encoding a ubiquitous isoform, ant and the failure to identify co-regulated genes with models built from its promoter region allow us to validate our strategy. moreover, this analysis of an unregulated ubiquitous gene becomes a standard for further works by providing a precise scale of similarity of matrices in our models. thus, a strategy of promotology, based simultaneously on a conclusive phylogenetic analysis and on already validated regulatory nucleotide sequences, allows for the identification of co-regulated genes. our work on this set of isoforms also showed that transcriptional regulation is a major mechanism of cellular specificity. the structure of the promoter sequence directly upstream of the transcription start site itself allows the identification of co-regulated genes. this suggests that, at least for the regulation of bioenergetic pathways described in this work, other supposed regulatory mechanisms including microrna or the messenger rna stability, would intervene in other cellular functions. moreover, our strategy allows overcoming the insufficiencies of other techniques used for the study of gene expression. taking into account the very close coding sequences of the four ant isoforms, no currently commercial microarrays able to simultaneously and specifically quantify the four transcripts is available. in addition, the very hydrophobic properties of these proteins do not allow their identification by 2d electrophoresis and no specific antibody of each isoform is available.  in conclusion, our computational strategy on a set of four isoforms, known for their specific functions in cell bioenergetics, enabled us to develop a powerful analysis of gene promoter sequences. our analyses enabled us to identify an entire set of co-regulated genes, involved in the same cellular function. this study validates the major role of the proximal promoter region in tissue specificity and should provide, along with transcriptomics and metabolomics, assistance in developing cellular metabolic networks and the study of their regulatory pathways. 
moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks. we developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ant isoform. these models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. the analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. in higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. transcriptional regulationpromoter analysisregulatory modelsadenine nucleotide translocator each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. from their promoter sequence and from the phylogenetic evolution of these ant genes in mammals, we constructed combinations of specific regulatory elements.
 the analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. in higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. we developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. this strategy was tested on a set of four human genes encoding isoforms to of the mitochondrial adp/atp carrier ant. each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics.  from their promoter sequence and from the phylogenetic evolution of these ant genes in mammals, we constructed combinations of specific regulatory elements. these models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. for each of transcriptionally regulated ant and genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases.  most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ant isoform. our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks. transcriptional regulationpromoter analysisregulatory modelsadenine nucleotide translocator 