the genes doublesex in drosophila and mab-3 in caenorhabditie elegans were first identified as transcription factors containing a conserved zinc finger - like dna binding domain ( dm domain ) , which plays a key role in sexual development 1 - 3 .
many organisms have multiple dmrt ( doublesex and mab-3 related transcriptional factor ) genes , the number of which varies between species . for example
drosophila , caenorhabditis elegans , fish , mice and human have four , eleven , six , seven and eight copies respectively 4 - 9 . from arthropods to nematodes to vertebrates , most dmrt genes are expressed in gonads and play roles in sex determination , which serves as a rare example of conserved mechanisms underlying sex regulation during evolution .
previous research with dmrt1-deficient mice strongly demonstrated that this gene was required for testis differentiation after determination , but dispensable for ovary development 11 . in birds , dmrt1 is sex - linked 12 , 13 on chromosome z and there is higher dosage of the gene in males ( zz ) as compared with females ( zw ) 10 , 13 - 15 .
the expression of dmrt1 in turtles and alligators was found to be related to sexual differentiation and was higher in developing male gonads than in female ones as well 16 .
furthermore , the dmrt1y / dmy gene , which is the homolog of dmrt1 in medaka , transposed to chromosome y and became a master gene in male differentiation 17 , 18 .
however , evidence is being accumulated that some proteins in the dmrt family are involved in non - gonadal development .
a null mutation for the gene dmrt1 , an ancient conserved gene common to sex - determining pathways , resulted in profound abnormalities in the development of anterior neural plate derivatives in ciona 19 . both dmrt2a and dmrt2b are expressed in presomitic mesoderm and developing somites and contribute to somitogenesis and the creation of left - right asymmetry in the lateral - plate mesoderm 20 - 23 . in chicken and mice ,
dmrt3 is expressed similarly in the forebrain , spinal cord , and nasal placode 24 , while dmrt3 in zebrafish is expressed in the olfactory placode and the neural tube 25 .
xenopus dmrt4 is expressed in the developing olfactory system and is required for neurogenesis 26 .
in addition to dmrt1 , dmrt7 expression is restricted to embryonic gonads and adult testis 9 .
dmrt7 deficient mice show male infertility with spermatogenic arrest at the pachytene stage and defects in regulation of sex chromatin 28 , 29 .
kawamata m , et al . reported a possible post transcriptional role of poly - adenylation in dmrt7 expression 30 .
however , the transcriptional mechanisms regulating dmrt7 need to be studied further , which will contribute substantially to our understanding of the pathway of sex differentiation and spermatogenesis . here
deletion and site - directed mutagenesis were used to identify regions of the promoter that are required for transcriptional activity .
emsa and chip analysis was employed to determine the binding relationship between transcription factor nf - y and the dmrt7 promoter .
finally , our study showed that nf - y up - regulated the expression of dmrt7 by binding to two tandem ccaat - boxes in the proximal promoter region of the gene .
in silico sequence analysis . transcription factor binding sites were predicted using matinspector ( www.genomatrix.de ) and tfsearch ( www.cbrc.jp/research/db/tfsearch.html ) .
six deletion mutants of the mouse dmrt7 promoter were constructed using pcr cloning from mouse genomic dna and primers : forward primer , 5'tatacgcgtctagaggtcacacacaaaatcagaag3 ' for construct -948/+116 , 5'tgcacgcgtaaaggcaggctaaaaagcctgcc3 ' for construct -407/+116 , 5'gggacgcgtcagcctcgctctggctgaggt3 ' for construct -245/+116 , 5'ttgacgcgtcggagaagcgggtaggcaagaaa3 ' for construct -104/+116 , 5'cggacgcgtattgcaaaccctattggctgcgc3 ' for construct -60/+116 , 5'ctcacgcgtccttgtgtgaaggagcgagcgg3 ' for construct + 1/+116 , and a common reverse primer , 5'cacaagcttcacagcctcgagccgaatcacag3 ' .
pcr products were double - digested with mlui and hindiii and cloned into the mlui - hindiii site of the pgl3-basic vector ( promega , madison , wi ) .
site - directed mutagenesis for two ccaat boxes was performed using the following primers as follows : mut1 for ccaat box1 mutant : 5'tgcaaaccctctgatctgcgcggcgccg3 ' and 5'cggcgccgcgcagatcagagggtttgca3 ' ; mut2 for ccaat box2 mutant , 5'gtgcttggagctcctgattccttgtgtg3 ' and 5'cacacaaggaatcaggagctccaagcac3 ' ; mut1/2 for both ccaat box 1 and 2 mutants using mut1 as a template : 5'gtgcttggagctcctgattccttgtgtg3 ' and 5'cacacaaggaatcaggagctccaagcac3 ' .
all constructs were sequenced . for the construction of expression plasmids , the full - length cdnas of mouse wild - type nf - ya ,
nf - yb and nf - yc were pcr - amplified from mouse testis cdnas and cloned into ecori site of the pcx - egfp vector by replacing the egfp fragment to generate expression plasmids ( pcx - nfya , pcx - nfyb and pcx - nfyc ) using primers : , 5cgtgaattcgccatggagcagtata3 and 5ctcgaattcttaggaaactcggatga3 for nf - ya ; 5atagaattcatcatgacaatggacgg3 and 5 tccgaattctcatgaaaactgaatttg3 for nf - yb ; 5gccgaattcaaaatgtccacagaag3 and 5ctcgaattctcagtctccagtcacct3 for nf - yc .
the mouse dominant negative plasmid for nf - ya ( nf - yam29 ) was a generous gift from dr .
gc-1 ( a cell line from type b spermatogonia of mouse and show characteristics of a stage between type b spermatogonia and primary spermatocytes ) and cos 7 cells ( a cell line was obtained by immortalizing a cv-1 cell line derived from kidney cells of the african green monkey ) were maintained in high glucose dulbeccos modified eagles medium supplemented with 10% fetal bovine serum , plated in 48-well plates and transfected using lipofectamine2000 ( invitrogen , carlsbad , ca , usa ) .
the cells were transfected with 0.4 g of each deletion construct ( -948/+116 , -407/+116 , -245/+116 , -104/+116 , -60/+116 , or + 1/+116 ) along with 10 ng / well prt - tk ( an internal control ) . for luciferase assays , 0.2 g of construct -60/+116 or mutated constructs , and 0.2 g nf - y expression vectors ( nf - ya , nf - yb and nf - yc ) or the dominant negative nf - y plasmid ( nf - yam29 ) were co - transfected into the cells .
empty vector pgl3-basic or pcx ( pcx - egfp without the egfp coding sequence between two ecori sites ) was added to equalize the final dna content in each well .
luciferase activity was measured 24 hours after transfection using the dual - luciferase reporter assay system ( promega , madison , wi , usa ) and a modulus single tube multimode reader ( turner biosystems , sunnyvale , ca , usa ) following the manufacturer 's protocol .
oligonucleotides corresponding to the ccaat box1 and box2 of the dmrt7 promoter were synthesized and annealed into double strands .
their sequences are as follows : 5tgcaaaccctattggctgcgcggcgccg3 and 5cggcgccgcgcagccaatagggtttgca3 for oligo1 ; 5tgcaaaccctctgatctgcgcggcgccg3 and 5cggcgccgcgcagatcagagggtttgca3 for oligo1-ccaat mut ; 5gtgcttggagctcattggtccttgtgtg3 and , 5cacacaaggaccaatgagctccaagcac3 for oligo2 ; 5gtgcttggagctcctgattccttgtgtg3 and 5cacacaaggaatcaggagctccaagcac3 for oligo2-ccaat mut .
radiolabeled probes were generated by incubation of 250 ng annealed oligonucleotides with 20 ci [ p ] datp in the presence of t4 polynucleotide kinase ( promega , madison , wi , usa ) for 1 h at 37c , and were subsequently separated from free nucleotides for purification using a g-50 column ( amersham biosciences , uppsala , sweden ) .
mouse testis nuclear extract used for electrophoretic mobility shift assays was prepared as described previously 31 .
then incubated at room temperature for 30 min with a 100,000 dpm radiolabeled probe and 1 g poly ( di - dc ) in a buffer of 10 mmol / l tris - hcl , ph 7.5 , 50 mmol / l nacl , 1 mmol / l dithiothreitol , 1 mmol / l edta , and 5% glycerol . for supershift experiments
, binding reactions were subsequently incubated with anti - nf - ya ( abcam inc . , cambridge , ca , usa ) for 30 min at room temperature . for competition experiments , the unlabeled competitor oligos ( in 50-fold molar excess )
samples were resolved in 5% polyacrylamide gels in 0.5% tbe running buffer at 10 v / cm for 2 h. the dried gel was exposed to a phosphorimager cassette and scanned with typhoon 9200 instrument ( ge - healthcare , amersham bioscience , uppsala , sweden ) .
( chip ) and quantitative real - time pcr . samples of mouse testis and liver were chopped into small pieces with a scalpel in cold phosphate - buffered saline ( pbs ) and cross - linked in 1% formaldehyde - pbs for 15 min with constant shaking .
the tissues were rinsed in cold pbs and homogenized with a dounce homogenizer in 1 ml cold cell lysis buffer ( 10 mm tris - cl , ph 8.0 , 10 mm nacl , 3 mm mgcl2 , 0.5% np-40 ) supplemented with protease inhibitors ( roche diagnostics ltd , mannheim , germany ) .
the cells were incubated at 4c for 5 min to allow the release of the nuclei .
after lysis in the buffer ( 1% sodium dodecyl sulfate [ sds ] , 5 mm edta , 50 mm tris - cl , ph 8.1 ) , sonication was performed with a sonic dismembrator model 100 sonicator ( fisher scientific , inc . ,
the supernatant chromatin after centrifugation was immunoprecipitated with no antibody ( beads only ) , preimmuno igg ( igg ) and anti - nf - ya ( abcam inc . ,
cambridge , ca , usa ) , together with protein g plus - agarose ( santa cruz , biotech , ca , usa ) respectively .
dna isolated from the immunoprecipitated complex was amplified by pcr with primers flanking both ccaat boxes .
the primer sequences are : 5agaagcgggtaggcaaga3 and 5cagtggcgaagaggaacg3. the amplified pcr fragments were analyzed on a 2% agarose gel .
a region of 257 bp around 10 kb downstream of the first exon of the dmrt7 was amplified as a control with primers : 5aataagtttccaaccgtgaagt3 and 5tatccaaggagatgggtaagtg3. the pcr products were cloned into t - easy vector ( promega , madison , wi , usa ) and sequenced for confirmation
. precipitated dna in the chip assay was amplified by quantitative real - time pcr using the multichannel rotorgene 3000 ( corbett research , australia ) .
the cycling conditions were : 5 min at 94c ; 45 cycles of 94c , 20 s ; 60c , 20 s and 72c , 15 s in a 25 l reaction mix containing sybr green i. the p1 region primers were 5agaagcgggtaggcaaga3 ; and 5cagtggcgaagaggaacg3. the p2 region primers were 5aataagtttccaaccgtgaagt3 ; and 5tatccaaggagatgggtaagtg3. each sample was performed in triplicate at least .
data were analyzed by the software rotor - gene version 4.6 and then plotted in microsoft excel .
aliquots of tissue extracts ( 50 mg ) were separated by electrophoresis in 10% sds - polyacrylamide gel ( page ) and transferred onto a 0.45 m membrane ( hybond - p ; amersham pharmacia biotech ) .
, san diego , ca ) and -actin was used as an internal control ( 1:1000 , santa cruz biotech , ca , usa ) .
membranes were incubated with primary antibody for 1 h at room temperature , washed in tbst and probed with hrp conjugated secondary antibody ( 1:10000 , santa cruz biotech , ca , usa ) .
after several washes in tbst , detection of the secondary antibody was performed using the supersignal chemiluminescent substrate system ( pierce , rockford , usa ) .
aliquots of tissue extracts ( 50 mg ) were separated by electrophoresis in 10% sds - polyacrylamide gel ( page ) and transferred onto a 0.45 m membrane ( hybond - p ; amersham pharmacia biotech ) .
dmrt7 was detected by dmrt7 antibody ( 1:500 , genway biotech , inc . , san diego , ca ) and -actin was used as an internal control ( 1:1000 , santa cruz biotech , ca , usa ) .
membranes were incubated with primary antibody for 1 h at room temperature , washed in tbst and probed with hrp conjugated secondary antibody ( 1:10000 , santa cruz biotech , ca , usa ) .
after several washes in tbst , detection of the secondary antibody was performed using the supersignal chemiluminescent substrate system ( pierce , rockford , usa ) .
to characterize functional elements in the dmrt7 promoter , the sequence from -407 to + 116 of dmrt7 was run in matinspector and tfsearch to find putative transcriptional elements .
several possible transcription - factor binding sites , which included ap-1 , sp1 , gata1 , e2f and ccaat box , were identified ( figure 1a ) .
blast alignment of the promoter region of mouse , rat , and human dmrt7 genes revealed two highly conserved ccaat boxes : the ccaat box1 ( -48/-44 ) and the ccaat box2 ( -7/-3 ) ( figure 1b ) . to determine the functional elements required for promoter activity ,
six 5'-deletion mutants ( -948/+116 , -407/+116 , -245/+116 , -104/+116 , -60/+116 and + 1/+116 ) were constructed and analyzed for promoter activity using luciferase reporter in gc-1 and cos 7 cells ( figure 1c ) .
the deletion of the region between -245 and -104 appears to increase promoter activity in gc-1 cells , whereas the opposite result was obtained in cos 7 cells . to explain this discrepancy , we speculate that there could be other gc-1 specific transcription factors that could bind to this region and play a negative role in dmrt7 promoter activity .
deletion of the -60 to + 1 sequence markedly decreased the activity , indicating the region of the -60 to + 1 is essential for promoter function ( figure 1c ) .
two highly conserved ccaat boxes were located in this region . to confirm the role of both ccaat boxes ,
the mutation of ccaat box1 in -60/+116 construct resulted in a decrease of approx.60 - 70% compared to the promoter activity of -60/+116 construct , the ccaat box2 mutation in -60/+116 construct approx.70 - 80% . nearly no activity was observed in the double mutations of both boxes in -60/+116 construct .
however , the double mutations of both ccaat boxes in the -948/+116 region resulted in only a decrease of approx.40 - 50% compared to the promoter activity of -60/+116 construct .
although there may be other important cis - regulatory sites upstream of the -60/+116 , which may compensate the loss of these ccaat boxes ( figure 1d ) , the region of the -60/+116 including two highly conserved ccaat boxes is important for the promoter function .
these results showed that both ccaat box1 and ccaat box2 are important for transcriptional activation of the mouse dmrt7 gene .
transcription factor prediction using the matinspector and tfsearch showed that potential transcription factor nf - y may bind to the ccaat boxes . to determine if nf - y binds to the ccaat boxes , electrophoretic mobility shift assays were carried out using nuclear extracts prepared from mouse testis and double stranded oligonucleotides .
incubation of probe1 spanning from -58 to -31 ( containing ccaat box1 ) with the extract gave rise to the formation of a dna - protein complex ( figure 2 lane1 ) , which could be competed by the addition of an excessive amount of unlabelled oligo1 dna ( figure 2 lane5 ) , but not by the oligo1 dna with mutated ccaat box ( figure 2 lane7 ) .
furthermore , addition of nfya antibody to the binding reaction caused the disappearance of the specific dna / protein complex and the appearance of the super - shift band ( figure 2 lane3 ) .
these results indicate that the ccaat box1 within the promoter region is capable of binding with transcription factor nf - ya in vitro .
a similar result was also observed in incubation of probe2 spanning from -20 to + 8 ( containing ccaat box2 ) with mouse testis nuclear extract ( figure 2 ) .
these results indicate that transcription factor nf - ya can bind to dmrt7 promoter in vitro . to further determine whether nf - y binds to the mouse dmrt7 promoter in vivo , we performed chromatin immunoprecipitation analysis . because dmrt7 protein was only detected in testis ( figure 3a ) , we chose the testis for chip analysis .
as shown in figure 3b , a 160 bp of dna fragment was amplified from the precipitate by nf - ya antibody from testis , but not from control tissue liver or negative control immunoprecipitations using no antibody ( beads only ) or normal rabbit igg .
the amplified fragment was confirmed by sequencing . to rule out the possibility of non - specific binding of the nf - ya antibody , an additional pcr amplification of a distinct genomic region
was performed on all of the precipitated chromatin dnas , which showed no amplification out of the nf - ya - binding region ( figure 3b , d ) .
a quantitative chip pcr was also performed to quantify the binding extent of nf - ya on the dmrt7 promoter ( figure 3c ) .
these results indicate that nf - y specifically binds to the dmrt7 promoter in vivo . to investigate the role of transcription factor nf - y in the activation of the dmrt7 promoter , both gc-1 and cos 7 cells were co - transfected with luciferase reporter driving by wild - type dmrt7 promoter ( -60/+116 construct ) or its mutants for ccaat sites , and nf - ya / b / c expression plasmids .
the luciferase activity increased obviously when a wild - type promoter construct ( -60/+116 ) was co - transfected with nf - ya / b / c ( figure 4a ) .
however , the luciferase activity was not upregulated obviously when co - transfected with mutants for ccaat box1 or box2 and was not upregulated absolutely when co - transfected with double mutants for ccaat box1 and box2 ( figure 4a ) .
this result indicated tandem arrangement of the ccaat box1 and box2 may ensure maximum promoter activity of the dmrt7 .
in addition , replacing the nf - ya / b / c expression plasmid with the dominant - negative construct ( nf - yam29 ) resulted in a marked reduction of luciferase activity in a dosage - dependent manner ( figure 4b ) .
nf - yam29 has three amino acid substitutions at the c - terminal region of the nf - ya , which impairs its dna binding and acts as a dominant - negative mutant by sequestering the nf - yb / yc subunits into a defective complex 32 .
to characterize functional elements in the dmrt7 promoter , the sequence from -407 to + 116 of dmrt7 was run in matinspector and tfsearch to find putative transcriptional elements .
several possible transcription - factor binding sites , which included ap-1 , sp1 , gata1 , e2f and ccaat box , were identified ( figure 1a ) .
blast alignment of the promoter region of mouse , rat , and human dmrt7 genes revealed two highly conserved ccaat boxes : the ccaat box1 ( -48/-44 ) and the ccaat box2 ( -7/-3 ) ( figure 1b ) . to determine the functional elements required for promoter activity ,
six 5'-deletion mutants ( -948/+116 , -407/+116 , -245/+116 , -104/+116 , -60/+116 and + 1/+116 ) were constructed and analyzed for promoter activity using luciferase reporter in gc-1 and cos 7 cells ( figure 1c ) .
the deletion of the region between -245 and -104 appears to increase promoter activity in gc-1 cells , whereas the opposite result was obtained in cos 7 cells . to explain this discrepancy , we speculate that there could be other gc-1 specific transcription factors that could bind to this region and play a negative role in dmrt7 promoter activity .
deletion of the -60 to + 1 sequence markedly decreased the activity , indicating the region of the -60 to + 1 is essential for promoter function ( figure 1c ) .
two highly conserved ccaat boxes were located in this region . to confirm the role of both ccaat boxes ,
the mutation of ccaat box1 in -60/+116 construct resulted in a decrease of approx.60 - 70% compared to the promoter activity of -60/+116 construct , the ccaat box2 mutation in -60/+116 construct approx.70 - 80% . nearly no activity was observed in the double mutations of both boxes in -60/+116 construct .
however , the double mutations of both ccaat boxes in the -948/+116 region resulted in only a decrease of approx.40 - 50% compared to the promoter activity of -60/+116 construct .
although there may be other important cis - regulatory sites upstream of the -60/+116 , which may compensate the loss of these ccaat boxes ( figure 1d ) , the region of the -60/+116 including two highly conserved ccaat boxes is important for the promoter function .
these results showed that both ccaat box1 and ccaat box2 are important for transcriptional activation of the mouse dmrt7 gene .
transcription factor prediction using the matinspector and tfsearch showed that potential transcription factor nf - y may bind to the ccaat boxes . to determine if nf - y binds to the ccaat boxes , electrophoretic mobility shift assays were carried out using nuclear extracts prepared from mouse testis and double stranded oligonucleotides .
incubation of probe1 spanning from -58 to -31 ( containing ccaat box1 ) with the extract gave rise to the formation of a dna - protein complex ( figure 2 lane1 ) , which could be competed by the addition of an excessive amount of unlabelled oligo1 dna ( figure 2 lane5 ) , but not by the oligo1 dna with mutated ccaat box ( figure 2 lane7 ) .
furthermore , addition of nfya antibody to the binding reaction caused the disappearance of the specific dna / protein complex and the appearance of the super - shift band ( figure 2 lane3 ) .
these results indicate that the ccaat box1 within the promoter region is capable of binding with transcription factor nf - ya in vitro .
a similar result was also observed in incubation of probe2 spanning from -20 to + 8 ( containing ccaat box2 ) with mouse testis nuclear extract ( figure 2 ) .
these results indicate that transcription factor nf - ya can bind to dmrt7 promoter in vitro . to further determine whether nf - y binds to the mouse dmrt7 promoter in vivo , we performed chromatin immunoprecipitation analysis . because dmrt7 protein was only detected in testis ( figure 3a ) , we chose the testis for chip analysis .
as shown in figure 3b , a 160 bp of dna fragment was amplified from the precipitate by nf - ya antibody from testis , but not from control tissue liver or negative control immunoprecipitations using no antibody ( beads only ) or normal rabbit igg .
the amplified fragment was confirmed by sequencing . to rule out the possibility of non - specific binding of the nf - ya antibody , an additional pcr amplification of a distinct genomic region
was performed on all of the precipitated chromatin dnas , which showed no amplification out of the nf - ya - binding region ( figure 3b , d ) .
a quantitative chip pcr was also performed to quantify the binding extent of nf - ya on the dmrt7 promoter ( figure 3c ) .
these results indicate that nf - y specifically binds to the dmrt7 promoter in vivo .
to investigate the role of transcription factor nf - y in the activation of the dmrt7 promoter , both gc-1 and cos 7 cells were co - transfected with luciferase reporter driving by wild - type dmrt7 promoter ( -60/+116 construct ) or its mutants for ccaat sites , and nf - ya / b / c expression plasmids .
the luciferase activity increased obviously when a wild - type promoter construct ( -60/+116 ) was co - transfected with nf - ya / b / c ( figure 4a ) .
however , the luciferase activity was not upregulated obviously when co - transfected with mutants for ccaat box1 or box2 and was not upregulated absolutely when co - transfected with double mutants for ccaat box1 and box2 ( figure 4a ) .
this result indicated tandem arrangement of the ccaat box1 and box2 may ensure maximum promoter activity of the dmrt7 .
in addition , replacing the nf - ya / b / c expression plasmid with the dominant - negative construct ( nf - yam29 ) resulted in a marked reduction of luciferase activity in a dosage - dependent manner ( figure 4b ) .
nf - yam29 has three amino acid substitutions at the c - terminal region of the nf - ya , which impairs its dna binding and acts as a dominant - negative mutant by sequestering the nf - yb / yc subunits into a defective complex 32 .
dmrt7 is a member of the dm domain family of genes , which encodes proteins with a conserved dna binding dm domain .
in addition to dmrt1 , some dm domain proteins such as dmrt3 , dmrt4 and dmrt7 may be involved in sexual development .
dmrt7 mrna could be detected in testis and fetal ovary but not in other tissues 9 .
the expression of dmrt7 protein was detected in testis 29 , but not in the fetal ovary .
dmrt7 mutants were infertile with spermatogenic arrest at the pachytene stage , while the mutant females showed normal fertility 28 , 29 .
possible post transcriptional role of poly - adenylation in dmrt7 expression regulation was previously proposed 30 . because transcriptional regulation of the gene dmrt7 during spermatogenesis is an important process , in this study , we have explored promoter function of the gene and found that nuclear factor - y ( nf - y ) regulates transcription of mouse dmrt7 gene through binding to two conserved ccaat boxes in its proximal promoter region .
deletion analysis of the 5 ' flanking region of the mouse dmrt7 revealed crucial positive elements between -60 and + 1 , in which two evolutionarily conserved ccaat boxes ( the ccaat box1 and box2 ) were identified .
the conservation of transcription factor binding sites between orthologue gene promoters may indicate their highly significant roles in maintaining transcriptional regulation of dmrt7 gene .
further site - directed mutagenesis and functional analysis were performed to confirm their role in maintenance of promoter function of dmrt7 .
the results suggest that binding by nf - y to these two tandem ccaat boxes is important for regulation of the basal transcription of the dmrt7 gene in the mouse testis .
nf - y protein ( also known as cbf ) binds with high specificity to the ccaat sequence and is a ubiquitous heteromeric transcription factor 33 .
nf - y is a complex composed of three subunits : nf - ya ( cbf - b , hap2 in yeast ) , nf - yb ( cbf - a , hap3 ) and nf - yc ( cbf - c , hap5 ) , which are all required for dna - binding 34 - 36 . both nf - yb and nf - yc
have conserved histone folding motifs resembling h2b - h2a and their heterodimerization is essential for nf - ya association 33 , 37 .
nf - ya is a regulatory subunit of the trimeric complex , whose levels vary in different cell types and/or growth conditions 38 . it has been suggested that nf - y can gain access to its genomic locations even in the absence of methyl histone marks and then leads the positioning of methyl histone marks typical of active chromatin independently 39 .
a number of mammalian promoters have been shown to contain ccaat boxes and have been regulated by nf - y alone or together with other transcriptional factors 41 - 44 . in accordance with these results ,
the transcription factor nf - y may activate the mouse dmrt7 gene and play an important role in the regulation of dmrt7 expression in testis .
this , therefore , suggests that nf - y regulation of dmrt7 plays a functional role in spermatogenesis . in summary
, we have found two important ccaat boxes in the 5 ' flanking region of the mouse dmrt7 promoter .
nf - y is a major transcriptional activator of the dmrt7 gene through binding to these two ccaat boxes .
these results provide evidence of the regulatory mechanisms that control the expression in testis of the mouse dmrt7 gene and will form the basis for understanding mammalian spermatogenesis .
 dmrt7 , a member of the dmrt family of genes , is required for spermatogenesis . 
 however , promoter functions of the gene dmrt7 remain unknown . we have cloned and characterized the proximal promoter region of the mouse dmrt7 gene . 
 functional analysis of the 5 ' flanking region by sequential deletion mutations revealed crucial positive elements between -60 and + 1 , in which two highly conserved and tandem ccaat boxes : the ccaat box1 ( -48/-44 ) and the ccaat box2 ( -7/-3 ) are located . 
 site - directed mutagenesis studies demonstrated that both ccaat boxes are indispensable to the promoter activity . 
 electrophoretic mobility shift assays ( emsas ) and gel - supershift assays indicated that transcription factor nf - y binds to the promoter . 
 chromatin immunoprecipitation ( chip ) analysis demonstrated that nf - y interacts in vivo with the promoter of the dmrt7 gene in testis . 
 co - transfection and reporter analysis showed that over - expression of nf - ys increased transcription of the dmrt7-luc gene whereas expression of a dominant - negative nf - ya decreased the transcription . 
 this suggests that nf - y can activate the dmrt7 promoter . 
 these results provide evidence of a transcription regulatory mechanism that controls dmrt7 gene expression in mouse testis . 