BACKGROUND
hepatoma derived growth factor  is a nuclear protein with mitogenic activity  <cit> .
it is highly expressed in developing heart and fetal gut  <cit> ; and re-expressed in vascular smooth muscle cells in vivo after vascular injury  <cit> , suggesting that it plays an important role in cardiovascular growth and differentiation.
recently by a number of investigators, hdgf was found to be tumorigenic and a prognostic factor for a number of cancers  <cit> .
we have discovered that hdgf functions as a transcriptional repressor, suggesting that hdgf may physiologically regulate cellular proliferation and differentiation by repressing the genes governing terminal differentiation .
however using nmr structural analyses, it is unclear if hdgf is a direct dna binding protein  <cit>  and controversial if the conserved n-terminal pwwp domain in hdgf is involved in dna binding  <cit> .
in the present study, using a modified chromatin immunoprecipitation  assay, we found that hdgf bound a conserved ~ <dig> base pair sequence common to an hdgf target gene promoter.
emsa and dnase i footprinting analysis revealed that hdgf-dna interaction is specific.
in emsa mapping studies, we found hdgf bound a minimal  <dig> bp long oligonucleotide and dna binding required the hdgf n-terminal pwwp domain.
furthermore, this dna sequence is functionally significant as hdgf represses a reporter gene which was controlled by the identified dna element.
RESULTS
hdgf binds to dna
hdgf is a nuclear protein  <cit>  and we recently have shown that it functions as a transcriptional repressor .
however, whether hdgf binds dna directly and if so, it's dna binding specificity are both unknown.
to functionally identify the hdgf dna binding sequence we used a modified chip assay to identify homologous sequences, if any, as potential dna binding motifs.
after  <dig> clones were sequenced, sequence alignment analysis found that there was a group of  <dig> sequences that shared a ~ <dig> bp highly conserved sequence .
no homologous sequences were identified in  <dig> clones which were immunoprecipitated by a gfp control antibody.
a highly conserved copy was found within the promoter of smyd <dig>  a hdgf target gene through database searching of the human genome .
we found this conserved element located at the promoter - <dig> to - <dig> bp proximal to the start codon.
alignments of the chip sequences with the smyd <dig> promoter identified a highly conserved  <dig> bp motif .
by chip-pcr, we successfully amplified this specific region  from immunoprecipitated chromatin dna using a hdgf specific antibody .
as a control, no amplification was observed for a proximal site .
we then verified hdgf dna binding using an in vitro competition emsa assay.
as shown in figure 2b, the labeled  <dig> bp probe bound tightly to gst-hdgf.
the binding was specific as  <dig> fold of the non-labeled  <dig> bp probe totally abolished the binding; however  <dig> fold of a nonspecific oligonucleotide competitor had no effect on the binding.
salmon sperm dna is known as a strong competitor for dna binding.
however, as shown in figure 2c, the binding of the  <dig> bp probe to hdgf could be blocked only when the concentration of salmon sperm dna reached  <dig> ug, which is approximately a  <dig>  molar fold excess to the input labeled oligo .
in addition, hdgf binding was not an artifact of using gst tagged hdgf as an emsa using another tagged protein, his-hdgf, or non-tagged in vitro translated hdgf, all demonstrated equivalent tight binding to the emsa probe .
from these results, we conclude that hdgf binds an  <dig> bp dna sequence conserved within the promoter of the hdgf target gene smyd <dig>
hdgf binds a  <dig> bp long dna motif specifically
to determine the hdgf core binding sequence, we made serial deletion oligonucleotides from the 3' end of the  <dig> bp probe, which we tested by emsa for hdgf binding.
as shown in figure 3a, the binding was completely lost with a probe shorter than  <dig> bp.
to further narrow down the dna recognition motif, we first took out another  <dig> bp at the 3' end of this  <dig> bp probe in figure 3a, then made another serial deletion set from the 5' end based on the  <dig> bp probe.
as shown in figure 3b, incremental deletion of the 5' end of the  <dig> bp probe, identified a  <dig> bp motif that is required for hdgf binding.
a  <dig> bp dna motif required for dna binding is relatively long.
in fact we could not detect any hdgf dna binding when we used  <dig> short dna probes which are all  <dig> bp long with a  <dig> bp overlap, covering the central part of the  <dig> bp oligo .
we also performed dnase i footprinting assay to determine the specificity of hdgf-dna interaction.
as shown in figure  <dig>  gst-hdgf can protect part of the  <dig> bp dna fragment from dnase i digestion.
the dna fragment used in footprinting analysis is the same as the probe used in emsa.
the protected region starts from  <dig> bp from the 3' end  to 5' end.
we could not get a clear footprint of 5' end because it is too close to the 5' of the dna fragment we used.
however, the result is consistent with the data of emsa, that the 5' of the  <dig> bp dna fragment is critical for hdgf-dna interaction.
these data indicated that hdgf binds dna specifically, but the binding requires a large dna molecule.
overexpression of hdgf represses a reporter gene when controlled by the hdgf binding element
in order to study whether the hdgf binding element in smyd <dig> gene promoter is functional, we cloned the  <dig> bp oligo described above upstream of the sv <dig> promoter, so that the hdgf binding element -sv <dig> controls the downstream luciferase gene.
co-transfection of hdgf with this reporter  significantly repressed the reporter gene activity in a dose dependent manner .
as a control, overexpression of hdgf had no effect on the reporter without the hdgf binding element .
these experiments indicate that hdgf exerts its repressive effect through binding with the hdgf binding element directly.
pwwp domain is required for dna binding
a pwwp domain spanning approximately  <dig> aa is present in the n-terminal of hdgf  <cit> .
although controversial, the pwwp domain is reported to have the capability of binding dna as has been shown for dna methyltransferase 3b   <cit> .
in order to study whether the hdgf pwwp domain is responsible for dna binding, we made several truncated hdgf fusion proteins to test for dna binding in emsa experiments.
as shown in figure  <dig>  only the truncated proteins which contain the whole pwwp domain can bind to dna , whereas proteins lacking the complete pwwp domain can not bind to dna.
importantly the entire pwwp domain is required as fusions containing a portion of the pwwp domain could not bind  or bound weakly .
discussion
hdgf is nuclear target protein with nuclear localization necessary for its mitogenic function  <cit> .
because of these attributes, it has been hypothesized that hdgf, especially considering it contains a pwwp domain, is a dna regulatory protein.
in this study, we found that hdgf binds directly to dna via a specific element contained in the promoter of a hdgf target gene, smyd <dig>  functionally this dna binding element also confers transcriptional repression activity.
smyd <dig> gene is essential for cardiomyocyte differentiation and cardiac morphogenesis  <cit> , the regulation of smyd <dig> by hdgf is in accord with the functional importance of hdgf in the development of cardiovascular system  <cit> , likely playing a role in preventing terminal differentiation.
we found that hdgf prefers to bind a large dna molecule, requiring a minimum  <dig> bp dna fragment.
it is surprising that hdgf binds such a long dna fragment.
in a recent structural study on the interaction of the hdgf pwwp domain with dna, we identified unique alterations in the structure of the hdgf pwwp domain with the addition of dna.
however we could not confirm whether these interactions with dna were specific  <cit> .
this was due to the technical requirements of both saab and nmr for oligonucleotides shorter than  <dig> bp.
in the present study, with emsa and longer oligonucleotides we found that the length of dna fragment is critical for hdgf binding.
hdgf tightly binds dna only when the dna is ≥ <dig> bp, suggesting that this hdgf/dna interaction may require a unique dna structure.
transcription factor binding sites are usually short, highly degenerate, and often require specific tertiary structures  <cit> .
for example estrogen receptor response element  is a palindromic motif.
however when we analyzed the  <dig> bp core sequence for hdgf binding using extractfeat program  <cit> , we could not identify any special structure such as palindromes or repeated structures within this  <dig> bp core hdgf binding sequence.
it is of interest that the  <dig> bp hdgf dna binding element is a component of an alu dna repeat.
the alu dna family is the most abundant members of the short interspersed repeated dna elements  which are found in the genomes of primates exclusively  <cit> .
alu sequences are approximately  <dig> bp in length, have a bipartite structure with left and right arm monomers which are derived from the 7sl rna gene.
the overall function of these repeats is unclear, but of interest to the present study, these repeats containing the hdgf binding sequence are found within the promoters of all hdgf target genes identified so far .
hdgf is evolutionary relative new.
besides mammals, hdgf homologs also exist in other organisms, such as xenopus and rodents.
although alu dna is found exclusively in primates, there is homologous sine dna in rodents such as the b <dig> and b <dig> dna elements of the mouse.
more interestingly, the mouse genome is estimated to contain  <dig>  copies of a retroposon family that is closely related to human alu repeats  <cit> .
comparative study of the dna binding in both human and mouse will give us more information on the conservation of this binding element.
the n-terminus of hdgf contains a pwwp domain, a weakly conserved  <dig> amino acid motif, originally identified in the whsc <dig> gene  <cit>  and as the hath  region in the hdgf family of proteins  <cit> , it has been found in more than  <dig> eukaryotic proteins  <cit> .
functionally, most of the pwwp family proteins are involved in chromatin remodeling  <cit> .
however the role of the pwwp domain in this function is unknown.
we demonstrate the first functional significance of the pwwp domain as it is necessary and sufficient for binding to the hdgf dna binding element.
the pwwp domain was hypothesized to be a site for protein-protein interactions  <cit> .
however, the pwwp domain of dnmt3b was shown to interact with dna  <cit> .
in a more detailed investigation, the pwwp domain of dnmt3b was found to bind major satellite dna nonspecifically  <cit> .
however, the pwwp in its closest homologue gene, dnmt3a  had no dna binding activity  <cit> .
this suggests that small changes in the pwwp domain may have a significant impact on its dna binding ability.
as the pwwp domain in hdgf is only  <dig> % homologous with dnmt3a, it is not surprising that hdgf could have a different dna binding behavior.
a recent structural study found that under physiological conditions, both pwwp modules and full length human hdgf could form dimers  <cit> .
using the spr method, the authors found that pwwp dimer binds to heparin with higher affinity than that of a monomeric pwwp module.
several sequence specific dna binding protein such as c/ebp, c-myc, c-jun and fos form hetero/homo dimers.
their leucine-zipper motifs do not face with the dna.
rather, they form three-dimensional "scaf-carboxylfolds" that match the contour of dna  <cit> .
whether hdgf dimerizes on dna and utilizes the similar mechanism of dna recognition remains to be determined.
CONCLUSIONS
we have shown that hdgf is a dna binding protein; specifically binds a dna element in the promoter of smyd <dig> gene.
this dna/hdgf interaction is unique in that the minimum required binding element was  <dig> bp in length.
importantly the poorly understood n-terminal pwwp domain of hdgf was responsible for dna binding.
taken together hdgf functions in the nucleus as a direct dna binding protein to repress the expression of specific target genes to likely regulate cell proliferation and differentiation.

 hepatoma derived growth factor is a nuclear protein with mitogenic activity. it is highly expressed in developing heart and fetal gut; and re-expressed in vascular smooth muscle cells in vivo after vascular injury, suggesting that it plays an important role in cardiovascular growth and differentiation. recently by a number of investigators, hdgf was found to be tumorigenic and a prognostic factor for a number of cancers. we have discovered that hdgf functions as a transcriptional repressor, suggesting that hdgf may physiologically regulate cellular proliferation and differentiation by repressing the genes governing terminal differentiation. however using nmr structural analyses, it is unclear if hdgf is a direct dna binding protein and controversial if the conserved n-terminal pwwp domain in hdgf is involved in dna binding. in the present study, using a modified chromatin immunoprecipitation assay, we found that hdgf bound a conserved ~ base pair sequence common to an hdgf target gene promoter. emsa and dnase i footprinting analysis revealed that hdgf-dna interaction is specific. in emsa mapping studies, we found hdgf bound a minimal bp long oligonucleotide and dna binding required the hdgf n-terminal pwwp domain. furthermore, this dna sequence is functionally significant as hdgf represses a reporter gene which was controlled by the identified dna element.  hdgf binds to dna hdgf is a nuclear protein and we recently have shown that it functions as a transcriptional repressor. however, whether hdgf binds dna directly and if so, it's dna binding specificity are both unknown. to functionally identify the hdgf dna binding sequence we used a modified chip assay to identify homologous sequences, if any, as potential dna binding motifs. after clones were sequenced, sequence alignment analysis found that there was a group of sequences that shared a ~ bp highly conserved sequence. no homologous sequences were identified in clones which were immunoprecipitated by a gfp control antibody. a highly conserved copy was found within the promoter of smyd a hdgf target gene through database searching of the human genome. we found this conserved element located at the promoter - to - bp proximal to the start codon. alignments of the chip sequences with the smyd promoter identified a highly conserved bp motif. by chip-pcr, we successfully amplified this specific region from immunoprecipitated chromatin dna using a hdgf specific antibody. as a control, no amplification was observed for a proximal site. we then verified hdgf dna binding using an in vitro competition emsa assay. as shown in figure 2b, the labeled bp probe bound tightly to gst-hdgf. the binding was specific as fold of the non-labeled bp probe totally abolished the binding; however fold of a nonspecific oligonucleotide competitor had no effect on the binding. salmon sperm dna is known as a strong competitor for dna binding. however, as shown in figure 2c, the binding of the bp probe to hdgf could be blocked only when the concentration of salmon sperm dna reached ug, which is approximately a molar fold excess to the input labeled oligo. in addition, hdgf binding was not an artifact of using gst tagged hdgf as an emsa using another tagged protein, his-hdgf, or non-tagged in vitro translated hdgf, all demonstrated equivalent tight binding to the emsa probe. from these results, we conclude that hdgf binds an bp dna sequence conserved within the promoter of the hdgf target gene smyd hdgf binds a bp long dna motif specifically to determine the hdgf core binding sequence, we made serial deletion oligonucleotides from the 3' end of the bp probe, which we tested by emsa for hdgf binding. as shown in figure 3a, the binding was completely lost with a probe shorter than bp. to further narrow down the dna recognition motif, we first took out another bp at the 3' end of this bp probe in figure 3a, then made another serial deletion set from the 5' end based on the bp probe. as shown in figure 3b, incremental deletion of the 5' end of the bp probe, identified a bp motif that is required for hdgf binding. a bp dna motif required for dna binding is relatively long. in fact we could not detect any hdgf dna binding when we used short dna probes which are all bp long with a bp overlap, covering the central part of the bp oligo. we also performed dnase i footprinting assay to determine the specificity of hdgf-dna interaction. as shown in figure gst-hdgf can protect part of the bp dna fragment from dnase i digestion. the dna fragment used in footprinting analysis is the same as the probe used in emsa. the protected region starts from bp from the 3' end to 5' end. we could not get a clear footprint of 5' end because it is too close to the 5' of the dna fragment we used. however, the result is consistent with the data of emsa, that the 5' of the bp dna fragment is critical for hdgf-dna interaction. these data indicated that hdgf binds dna specifically, but the binding requires a large dna molecule. overexpression of hdgf represses a reporter gene when controlled by the hdgf binding element in order to study whether the hdgf binding element in smyd gene promoter is functional, we cloned the bp oligo described above upstream of the sv promoter, so that the hdgf binding element -sv controls the downstream luciferase gene. co-transfection of hdgf with this reporter significantly repressed the reporter gene activity in a dose dependent manner. as a control, overexpression of hdgf had no effect on the reporter without the hdgf binding element. these experiments indicate that hdgf exerts its repressive effect through binding with the hdgf binding element directly. pwwp domain is required for dna binding a pwwp domain spanning approximately aa is present in the n-terminal of hdgf. although controversial, the pwwp domain is reported to have the capability of binding dna as has been shown for dna methyltransferase 3b. in order to study whether the hdgf pwwp domain is responsible for dna binding, we made several truncated hdgf fusion proteins to test for dna binding in emsa experiments. as shown in figure only the truncated proteins which contain the whole pwwp domain can bind to dna, whereas proteins lacking the complete pwwp domain can not bind to dna. importantly the entire pwwp domain is required as fusions containing a portion of the pwwp domain could not bind or bound weakly. discussion hdgf is nuclear target protein with nuclear localization necessary for its mitogenic function. because of these attributes, it has been hypothesized that hdgf, especially considering it contains a pwwp domain, is a dna regulatory protein. in this study, we found that hdgf binds directly to dna via a specific element contained in the promoter of a hdgf target gene, smyd functionally this dna binding element also confers transcriptional repression activity. smyd gene is essential for cardiomyocyte differentiation and cardiac morphogenesis, the regulation of smyd by hdgf is in accord with the functional importance of hdgf in the development of cardiovascular system, likely playing a role in preventing terminal differentiation. we found that hdgf prefers to bind a large dna molecule, requiring a minimum bp dna fragment. it is surprising that hdgf binds such a long dna fragment. in a recent structural study on the interaction of the hdgf pwwp domain with dna, we identified unique alterations in the structure of the hdgf pwwp domain with the addition of dna. however we could not confirm whether these interactions with dna were specific. this was due to the technical requirements of both saab and nmr for oligonucleotides shorter than bp. in the present study, with emsa and longer oligonucleotides we found that the length of dna fragment is critical for hdgf binding. hdgf tightly binds dna only when the dna is ≥ bp, suggesting that this hdgf/dna interaction may require a unique dna structure. transcription factor binding sites are usually short, highly degenerate, and often require specific tertiary structures. for example estrogen receptor response element is a palindromic motif. however when we analyzed the bp core sequence for hdgf binding using extractfeat program, we could not identify any special structure such as palindromes or repeated structures within this bp core hdgf binding sequence. it is of interest that the bp hdgf dna binding element is a component of an alu dna repeat. the alu dna family is the most abundant members of the short interspersed repeated dna elements which are found in the genomes of primates exclusively. alu sequences are approximately bp in length, have a bipartite structure with left and right arm monomers which are derived from the 7sl rna gene. the overall function of these repeats is unclear, but of interest to the present study, these repeats containing the hdgf binding sequence are found within the promoters of all hdgf target genes identified so far. hdgf is evolutionary relative new. besides mammals, hdgf homologs also exist in other organisms, such as xenopus and rodents. although alu dna is found exclusively in primates, there is homologous sine dna in rodents such as the b and b dna elements of the mouse. more interestingly, the mouse genome is estimated to contain copies of a retroposon family that is closely related to human alu repeats. comparative study of the dna binding in both human and mouse will give us more information on the conservation of this binding element. the n-terminus of hdgf contains a pwwp domain, a weakly conserved amino acid motif, originally identified in the whsc gene and as the hath region in the hdgf family of proteins, it has been found in more than eukaryotic proteins. functionally, most of the pwwp family proteins are involved in chromatin remodeling. however the role of the pwwp domain in this function is unknown. we demonstrate the first functional significance of the pwwp domain as it is necessary and sufficient for binding to the hdgf dna binding element. the pwwp domain was hypothesized to be a site for protein-protein interactions. however, the pwwp domain of dnmt3b was shown to interact with dna. in a more detailed investigation, the pwwp domain of dnmt3b was found to bind major satellite dna nonspecifically. however, the pwwp in its closest homologue gene, dnmt3a had no dna binding activity. this suggests that small changes in the pwwp domain may have a significant impact on its dna binding ability. as the pwwp domain in hdgf is only % homologous with dnmt3a, it is not surprising that hdgf could have a different dna binding behavior. a recent structural study found that under physiological conditions, both pwwp modules and full length human hdgf could form dimers. using the spr method, the authors found that pwwp dimer binds to heparin with higher affinity than that of a monomeric pwwp module. several sequence specific dna binding protein such as c/ebp, c-myc, c-jun and fos form hetero/homo dimers. their leucine-zipper motifs do not face with the dna. rather, they form three-dimensional "scaf-carboxylfolds" that match the contour of dna. whether hdgf dimerizes on dna and utilizes the similar mechanism of dna recognition remains to be determined.  we have shown that hdgf is a dna binding protein; specifically binds a dna element in the promoter of smyd gene. this dna/hdgf interaction is unique in that the minimum required binding element was bp in length. importantly the poorly understood n-terminal pwwp domain of hdgf was responsible for dna binding. taken together hdgf functions in the nucleus as a direct dna binding protein to repress the expression of specific target genes to likely regulate cell proliferation and differentiation. 
functionally overexpression of hdgf represses a reporter gene which is controlled by an sv- promoter containing the bp dna element. hdgf exerts its transcription repressive effect through binding to a conserved dna element in the promoter of target genes. moreover, chip and dnase i footprinting analysis revealed that hdgf binds this bp dna fragment specifically. recently we demonstrated that hdgf is a transcriptional repressor, but whether hdgf binds dna, the specificity of dna binding and what protein domain is required are still unknown. hepatoma derived growth factor is a nuclear protein with nuclear targeting required for mitogenic activity. homology analysis identified the binding sequences as a motif within the promoter of the smyd gene, a hdgf target gene. hdgf is a dna binding protein, binds dna specifically, and prefers a minimum of bp long dna fragment. using serial truncations of gst-hdgf, we mapped the dna binding domain of hdgf to the n-terminal pwwp domain. the n-terminal pwwp domain of hdgf is required for dna binding.
 hepatoma derived growth factor is a nuclear protein with nuclear targeting required for mitogenic activity. recently we demonstrated that hdgf is a transcriptional repressor, but whether hdgf binds dna, the specificity of dna binding and what protein domain is required are still unknown. in this study, we aimed to identify if hdgf is a dna binding protein, map the functional dna binding domain and dna binding element for hdgf.  using chromatin immunoprecipitation of human dna, we isolated dna sequences sharing a conserved ~ bp element. homology analysis identified the binding sequences as a motif within the promoter of the smyd gene, a hdgf target gene. electrophoretic mobility shift assays confirmed the binding of hdgf to this conserved sequence. as a result, an bp conserved sequence located in the smyd promoter bound gst-hdgf tightly. the binding core sequence for hdgf was narrowed down to bp using a deletion mapping strategy from both the 5' and 3' ends. moreover, chip and dnase i footprinting analysis revealed that hdgf binds this bp dna fragment specifically. functionally overexpression of hdgf represses a reporter gene which is controlled by an sv- promoter containing the bp dna element. using serial truncations of gst-hdgf, we mapped the dna binding domain of hdgf to the n-terminal pwwp domain.  hdgf is a dna binding protein, binds dna specifically, and prefers a minimum of bp long dna fragment. the n-terminal pwwp domain of hdgf is required for dna binding. hdgf exerts its transcription repressive effect through binding to a conserved dna element in the promoter of target genes. 