Mastermind-like Domain-containing 1 (MAMLD1 or CXorf6) Transactivates the Hes3 Promoter, Augments Testosterone Production, and Contains the SF1 Target Sequence*

Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.

Chromosome X open reading frame 6 (CXorf6) 2 was identified by Laporte et al. (1,2) as a candidate gene for 46,XY disorders of sex development. It spans ϳ70 kb in genomic sequence and comprises at least seven exons. An open reading frame resides on exons 3-6 and produces two proteins of 701 and 660 amino acids because of in-frame alternative splicing with and without exon 4. PCR-based human cDNA library screening has revealed ubiquitous expression of both splice variants, with the exon 4 positive variant being the major form (3). To date, however, the products of CXorf6 have been poorly characterized, although glutamine-and proline-rich domains have been identified on exon 3 (1).
We have recently shown that CXorf6 is a causative gene for hypospadias (3), a common male external genital anomaly defined by the urethral opening on the ventral side of the penis and classified into several types on the basis of the anatomical location of the urethral meatus (4). This notion is based primarily on the identification of nonsense mutations in two maternally related half-brothers (E124X) and in two sporadic boys (Q197X and R653X) with penoscrotal hypospadias (3). Because the mouse homolog (G630014P10Rik, NM_001081354) is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development, it is likely that the CXorf6 mutations cause hypospadias primarily because of testicular dysfunction and the resultant compromised testosterone production around that period (3). Indeed, although various genetic and environmental factors have been implicated in the development of hypospadias, it has been widely accepted that hypospadias can be caused by impaired testosterone effects around the critical period for sex development (4). Furthermore, the mouse homolog is co-expressed with steroidogenic factor 1 (SF1; aliases, AD4BP and NR5A1) (3), which regulates the transcription of a vast array of genes involved in sex devel-opment (5-7), suggesting a possible interaction between SF1 and CXorf6.
Mastermind-like 2 (MAML2; alias, Mam-3) is a non-DNAbinding transcriptional co-activator in Notch signaling (8,9) that plays an important role in cell differentiation in multiple tissues by exerting either inductive or inhibiting effects according to the context of the cells (10). Upon ligand-receptor interaction, Notch intracellular domain (N-ICD) is translocated from the cell surface to the nucleus and interacts with a DNAbinding transcription factor, recombination signal binding protein-J (RBP-J), to activate target genes like hairy/enhancer of split 1 (Hes1) and Hes5 (11). In this canonical Notch signaling process, MAML2 forms a ternary complex with N-ICD and RBP-J at nuclear bodies, enhancing the transcription of the Notch target genes (8,9,(12)(13)(14).
However, not all Hes genes are activated by the canonical Notch signaling pathway (11,15,16). Among such a distinct class of Hes genes, recent studies have shown that Hes3 can be induced by stimulation with a Notch ligand, via a STAT3 (signal transducer and activator of transcription 3)-mediated pathway (17). This finding, together with the lack of Hes3 induction by N-ICD (16), implies that Hes3 represents a target gene of a noncanonical Notch signaling.
Here, we report that CXorf6 produces a protein that has a structural homology with MAML2 and transactivates the Hes3 promoter activity and that CXorf6 is involved in testosterone production and harbors an SF1 target sequence.

EXPERIMENTAL PROCEDURES
Structural Analysis of CXorf6 Protein-We searched BLAST and TBlastn data bases using the CXorf6 protein sequence (NP_005482) as a bait. Protein sequences for the CXorf6 orthologs were predicted by comparing the human CXorf6 sequence with the genomic and transcribed sequences of different organisms using Clustal_X (18). The unrooted phylogram was generated by Clustal_X (18) from the sequence alignment of CXorf6 proteins and was visualized using TreeView 1.6.6 (19).
Primers, Probes, and Small Interfering RNAs (siRNAs)-The sequences of primers, probes, and siRNAs utilized in this study are summarized in supplemental Table 1.
Plasmid Vectors Utilized for CXorf6 Analyses-The cDNAs of the full-length CXorf6 (amino acids 1-701) and the minor splicing variant lacking exon 4 (⌬Exon 4) were amplified from human fetal testis cDNA (Invitrogen) and subcloned into pEF-BOS vector (20) to construct the CXorf6 expression vector for the transactivation analysis. The expression vectors containing cDNAs of nonsense mutants and missense variants of CXorf6 were constructed by mutagenesis. For the subcellular localization analysis, cDNAs for the wild-type, mutant, and variant CXorf6 were designed to lose the start codon and fused to the C-terminal side of the gene encoding either red fluorescent protein (RFP) in pDsRED-monomer C1 vector or green fluorescent protein (GFP) in pAcGFP1-C1 vector (Clontech). For the Western blot analysis, cDNAs missing the start codon were subcloned into pCMV-Myc vector (Clontech).
Cell Culture-We primarily utilized mouse Leydig tumor (MLT) cells (ATCC, CRL-2065 TM ), which retain the capability to produce testosterone and the responsiveness to human chorionic gonadotropin (hCG) stimulation (24), because CXorf6 is a causative gene for hypospadias that is predicted to result from impairment of hCG-dependent testosterone production around the critical period for sex development (4,25) and because the mouse homolog for CXorf6 is expressed in testosterone-producing Leydig cells (3). We also utilized COS1 cells and HEK293 cells depending on the experimental purposes. These cells were maintained in RPMI 1640 or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
Transactivation Analysis of CXorf6-Transactivation function of CXorf6 was analyzed by the luciferase methods. MLT cells seeded in 6-well dishes (1.0 -1.5 ϫ 10 5 cells/well) were transiently transfected using Lipofectamine 2000 (Invitrogen) with 0.6 g of luciferase reporter vector, 0.6 g of expression vector for CXorf6 or MAML2, and/or 0.8 g of expression vector for N1-ICD or N2-ICD, together with 20 ng of pRL-CMV vector used as an internal control. As controls for the expression vectors, empty counterpart vectors were transfected. Luciferase assays performed with a Lumat LB9507 (Berthold) at 48 h after transfection were repeated 4 -5 times.
DNA Binding Analysis of CXorf6-We searched for conserved regions (CRs) between the mouse Hes3 promoter sequence in the pHes3-luc vector and the human AL031847 sequence containing HES3 and ϳ100.2 kb upstream and ϳ63.8 kb downstream regions using the BLAST data base and performed an electrophoretic mobility shift assay (EMSA) for the CRs using a Lightshift chemiluminescent EMSA kit (Pierce). The procedure was as described in the manufacturer's instructions. In short, MLT cells or COS1 cells cultured in a plate with a diameter of 10 cm were transfected with 5 g of empty or human CXorf6 cDNA positive vector, and nuclear extracts were obtained at 48 h after transfection. Then, a small amount of nuclear extracts was incubated with each of biotin-labeled 24 -35-bp probes (20 fmol) covering the CRs, and the incubation mixture was subjected to gel electrophoresis. Subsequently, the biotin-labeled probe was detected by chemiluminescence on a nylon membrane.
Western Blot Analysis of CXorf6-Expression vectors for various Myc-tagged CXorf6 proteins (5 g) were transfected into HEK293 cells in a plate with a diameter of 6 cm. Cell lysates obtained at 48 h after transfection were probed with antibodies for Myc and ␤-actin utilized as an internal control.
Subcellular Localization Analysis of CXorf6-Subcellular localization of CXorf6 proteins was studied by expressing fusion proteins with RFP or GFP. Vectors for fusion proteins (2 Functional Analysis of MAMLD1 (CXorf6) g) were transfected into MLT cells in a glass dish with a diameter of 3.5 cm. The fluorescent signals were observed at 48 -72 h after transfection using a laser-scanning microscope LSM510 (version 3.2; Carl Zeiss) shortly after nuclear staining with 4Ј,6-diamidino-2-phenylindole.
Nonsense-mediated mRNA Decay (NMD)-Reverse transcriptase (RT)-PCR was performed for two regions of CXorf6 using lymphoblastoid cell lines of the patient with R653X and his heterozygous mother, with and without the treatment of an NMD inhibitor cycloheximide (CHX) (Sigma; 100 g/ml, 8-h incubation) (26). The occurrence of NMD was assessed by the presence or absence of the PCR products on the agarose gel in the patient and by the heterozygosity or hemizygosity of the PCR products on the electrochromatograms (CEQ 8000 Autosequencer, Beckman Coulter) in the mother after demonstrating a random X-inactivation pattern by the previously described method (27). Furthermore, maternal RT-PCR products were subcloned with a TOPO TA cloning kit (Invitrogen); 100 clones were subjected to sequencing to confirm the stabilization of mRNA with a nonsense mutation after CHX treatment.
Expression Analysis of HES3/Hes3-Human cDNA samples of penile and genital skin fibroblasts were prepared by RT-PCR using tissues obtained, after receiving permission, from a prepubertal boy with phimosis and from a prepubertal patient with ambiguous genitalia and mutant androgen receptor gene. Other human cDNA samples were purchased from Invitrogen or Clontech. For mouse Hes3, RT-PCR was performed for the MLT cells.
Knockdown Analysis for Mouse CXorf6 Homolog-We performed transient knockdown assay for the mouse CXorf6 homolog using two siRNAs (siRNA1 and siRNA2; final concentration 20 nM). The siRNAs were transfected into MLT cells seeded in 12-well dishes (5 ϫ 10 4 cells in each well with 1 ml of culture medium, using Lipofectamine RNAimax (Invitrogen). A nontargeting RNA (4611G, Ambion) was similarly transfected as a negative control.
After 48 h of incubation, we the examined mRNA quantity of mouse CXorf6 homolog in the harvested MLT cells and testosterone concentration in the culture medium using half of the wells. The relative amount of mRNA was determined by the Taqman real-time PCR method using the probe-primer mix for mouse CXorf6 homolog (assay No. mm01293665_m1, ABI) on an ABI PRISM 7000, using ␤2-microglobulin for an endogenous control. Testosterone concentration was measured by an electrochemiluminescence immunoassay. In addition, we further analyzed the testosterone production potential of the siRNA-transfected MLT cells in the remaining wells. After changing the old medium with a fresh medium containing hCG (Mochida Pharmaceutical; final concentration, 50 IU/liter), we cultured the cells for a further 1 h and measured testosterone concentration in the medium. These siRNA experiments were performed three times.
SF1 Target Sequence in CXorf6-We searched for a putative SF1 binding site in the genomic sequences of CXorf6 (AC109994) and the mouse homolog (NT_039706) and performed DNA binding and the transactivation analyses. For the DNA binding analysis, the 35 S-labeled 30-bp probes containing the putative SF1 binding site in CXorf6 were incubated with nuclear extracts of COS1 cells transfected by an empty or human SF1 cDNA positive vector (pRK5) (Addgene) or with recombinant mouse Sf1 protein and were subjected to gel electrophoresis. Similar analysis was also performed for a 32-bp probe harboring the known SF1 binding site of CYP11A1 (28) as a control. Furthermore, the biotin-labeled 30-bp probes containing the wild-type or the mutated SF1 binding site were incubated with nuclear extracts of COS1 cells transfected by a human SF1 cDNA positive vector (pCMX-PL2) and were subjected to gel electrophoresis.
For the transactivation analysis, a fragment (Ϫ1,924 ϳ Ϫ1,690) containing a putative SF1 binding site of CXorf6 was PCR-amplified and inserted into the pGL3 basic luciferase reporter vector (Promega). Furthermore, a reporter vector carrying the mutation in the putative SF1 binding site was generated by mutagenesis. These reporter vectors (0.5 g) were transfected into the MLT cells together with the empty or human SF1 cDNA positive expression vector (2.5 g) as well as pRL-CMV vector (20 ng) used as an internal control. The luciferase assays were repeated three times.
Statistical Analysis-The results are expressed as the mean Ϯ S.D. and statistical significance was determined by the t test. p Ͻ 0.05 was considered significant.

RESULTS
Structural Analysis of CXorf6 Protein-We found that CXorf6 protein has a unique structure with homology to that of MAML2 protein (Fig. 1A). A unique amino acid sequence, which we designated the mastermind-like (MAML) motif, was inferred from sequence alignment with MAML1, MAML2, and MAML3 proteins (8,9). The MAML motif was well conserved among CXorf6 orthologs identified in frog, bird, and mammals (Fig. 1B). In addition, a serine-rich domain was identified in CXorf6, as well as glutamine-and proline-rich domains.
Transcriptional Transactivation by the Wild-type CXorf6 Protein-We examined whether the wild-type CXorf6 (with exon 4) protein is involved in Notch signaling ( Fig. 2A). Expression of CXorf6 alone slightly but significantly increased the luciferase activity in the absence of Hes promoters (pGL2 basic only), probably via some backbone vector sequence. This phenomenon was more evident for other vectors such as pGL3 basic and pGL4 basic (not shown). Thus, we utilized pGL2 basic-based luciferase reporter constructs with the promoter sequences of Hes1, Hes5, and Hes3 (16).
For the canonical Notch target genes Hes1 and Hes5 with the RBP-J binding site (16), CXorf6 was incapable of enhancing the promoter activities beyond those observed for the pGL2 basic only. MAML2 had no transactivating function, and N-ICDs activated the promoters. The N-ICD-induced promoter activities were further enhanced by CXorf6, probably because of additive or synergic effects via some backbone vector sequence, and by MAML2 because of its co-activator function. The results from the MAML2 and N-ICDs studies were consistent with those reported previously (8,9,16).
By contrast, for the noncanonical Notch target gene Hes3 without the RBP-J binding site (16), CXorf6 alone was capable of enhancing its promoter activity, whereas MAML2 and Functional Analysis of MAMLD1 (CXorf6) FEBRUARY 29, 2008 • VOLUME 283 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5527 N-ICDs had no transactivating function. Consistent with this, co-expression of N-ICDs and CXorf6 or MAML2 exhibited no additive or synergic effects on the promoter activity.
These results argue that CXorf6 exerts its transactivation activity independently of the RBP-J binding sites. To confirm this, we performed similar analysis using pTP1-luc, which possesses an iterated enhancer element with an RBP-J binding site (23). As expected, CXorf6 was incapable of enhancing the N-ICD-induced transactivation, whereas MAML2 augmented the N-ICD-induced activities of this promoter (Fig. 2B).
DNA Binding Analysis of the Wild-type CXorf6 Protein-We attempted to examine whether the wild-type CXorf6 can bind to the Hes3 promoter sequence directly (supplemental Fig. 1). Comparison of the 2,976-bp mouse Hes3 promoter sequence with the human AL031847 sequence identified five CRs (CR1-CR5). Notably, the five CRs found in the human also resided in the upstream of the coding sequences of HES3, and the orientation of the CR1-CR5 was well conserved between human and mouse, whereas the mouse Hes3 promoter region was associated with repeat sequences between CR4 and CR5. EMSA was carried out using 24 -35-bp overlapping biotinlabeled probes covering the CR1-CR5 (total, 25 probes), showing no evidence for the DNA-binding capacity of CXorf6.
Transactivation Function of Mutant and Variant CXorf6 Proteins-We next analyzed the transactivating activities of the previously identified three apparently pathologic nonsense mutants and three apparently nonpathologic missense variants of CXorf6 (3) (Fig. 3A) using the pHes3-luc vector. The E124X and Q197X proteins had no transactivation function, whereas the R653X protein as well as the three variant (P286S, Q507R, and N589S) proteins retained a nearly normal transactivating activity (Fig.  3B). In addition, the transactivation function was significantly reduced in the L103P protein (an artificially constructed variant affecting the MAML motif) and was normal in ⌬Exon 4. Transactivation analysis was also performed in the presence of N-ICDs, showing similar results (not shown). Western blot analysis for the three nonsense mutants verified the presence of proteins with expected molecular masses, whereas expression of the E124X protein appeared to be relatively reduced and that of Q197X protein was somewhat increased (Fig. 3C).

Subcellular Localization Analysis of Various CXorf6
Proteins-The RFP-CXorf6 (wild-type with exon 4) fusion protein were distributed in a speckled pattern and co-localized with the MAML2-GFP fusion protein (Fig. 4A). Furthermore, although the GFP-E124X and GFP-Q197X fusion proteins resided in the    nucleus, they were incapable of localizing to the nuclear bodies (Fig. 4B). The remaining fusion proteins formed between the GFP and CXorf6 mutants and variants including R653X showed a punctate pattern (Fig. 4B) and co-localized with the RFP-CXorf6 (wild-type with exon 4) fusion protein (not shown).
Nonsense-mediated mRNA Decay-The above results indicate that the artificially produced R653X protein retains an almost normal function. In this regard, we have shown that R653X, as well as E124X and Q197X, causes NMD in vivo, by RT-PCR analysis of leukocytes of the patients (3). Indeed, the positions of these mutations including R653X (1957C Ͼ T) satisfy the condition for the occurrence of NMD (29).
To further confirm this event in the R653X mutation, we examined two regions of CXorf6 mRNA obtained from lymphoblastoid cell lines of the patient and his heterozygous mother, using an NMD inhibitor, CHX (Fig. 5A). In the patient, RT-PCR amplification for regions 1 and 2 yielded no or very faint product without CHX treatment and a clear band with CHX treatment (Fig. 5B). In the mother, methylation pattern analysis of the androgen receptor gene (exon 1) indicated random X-inactivation (40:60%), and RT-PCR direct sequencing for a part of the region 2 encompassing the mutation delineated only the wild-type allele without CHX treatment and both wildtype and mutant alleles with CHX treatment (Fig. 5C). Furthermore, the analysis of 100 maternal RT-PCR clones showed only the wild-type sequence without CHX treatment and both wild-type and mutant sequences with a ratio of 56:44, which is similar to the X-inactivation ratio, after CHX treatment. These findings argue for the occurrence of NMD in the R653X mutation.
Expression Analysis of HES3/Hes3-PCR-based cDNA library screening for HES3 identified variable degrees of expression in a range of tissues including fetal testis and adult ovary (Fig. 6). In addition, Hes3 was expressed in the MLT cells (not shown).
Knockdown Analysis for Mouse CXorf6 Homolog-At 48 h after transfection, the mRNA level of the endogenous mouse CXorf6 homolog was severely reduced in the MLT cells (relative amount: 28% for siRNA1 and 29% for siRNA2), indicating the successful knockdown (Fig. 7A). At that time, testosterone concentration was also significantly decreased in the medium harboring the knockdown cells (relative concentration: 63% for siRNA1 and 81% for siRNA2) (Fig. 7B). Furthermore, hCGstimulated testosterone production during a subsequent 1 h was also compromised in the knockdown MLT cells (relative concentration: 53% for siRNA1 and 55% for siRNA2) (Fig. 7C).
SF1 Target Sequence in CXorf6-A putative SF1 binding sequence, CCAAGGTCA, was identified at intron 2 upstream of the coding region of CXorf6 (Ϫ1,773 ϳ Ϫ1,765) (Fig. 8A). This binding site was also found at intron 1 in the upstream coding region in the mouse homolog (Ϫ42,904 ϳ Ϫ42,896 and Ϫ9,986 ϳ Ϫ9,978) (not shown). Both human SF1 and mouse Sf1 proteins were capable of binding to the putative SF1 binding site of CXorf6 as well as to the known SF1 binding site of CYP11A1 (Fig. 8B). This SF1 protein binding was drastically reduced when the putative SF1 binding site CCAAGGTCA was mutated as CCATTGTCA (Fig. 8C). Consistent with this finding, although the SF1 protein transactivated the luciferase activity of the wild-type reporter, the transactivation function of SF1 protein was significantly reduced for the mutant reporter (Fig. 8C).

DISCUSSION
The wild-type CXorf6 co-localized with structurally related MAML2 in the nuclear bodies and transactivated the Hes3 promoter without demonstrable DNA-binding capacity. These findings are consistent with transcription usually occurring around nuclear bodies (30,31) and suggest that CXorf6 may be recruited to the Hes3 promoter as a non-DNA-binding transcriptional co-activator, although the possibility that CXorf6   protein can bind to a non-examined sequence(s) has not been excluded at present. It might be possible, therefore, that MAML2 and CXorf6 are distantly related molecules derived from a common ancestor and that MAML2 has evolved as a co-activator for the RBP-J dependent canonical Notch signaling, whereas CXorf6 has evolved as a co-activator for the transcription of noncanonical Notch target Hes3. In this regard, although MAML2 can augment the endogenous RBP-Jdependent transcription of canonical Notch target genes only in the presence of N-ICD (Refs. 8 and 9 and the present study), CXorf6 alone was capable of enhancing the Hes3 promoter activity. This would be relevant to CXorf6 being devoid of the N-terminal region of MAML2 including the basic domain, which is essential for the ternary complex formation of MAML2 with N-ICD and RBP-J at the nuclear bodies (8,9,(12)(13)(14), and implies that CXorf6 may directly interact with an unknown endogenous DNA-binding transcription factor for Hes3. Although STAT3 may be a candidate DNA-binding transcription factor for Hes3 (17), there have been no data indicating a STAT3 binding to the Hes3 promoter in the noncanonical Notch signaling pathway, and STAT3 is expressed predominantly in the germ cells rather than in the CXorf6 expression positive Sertoli and Leydig cells in the developing testis (32). Thus, further studies are required to clarify how CXorf6 transactivates Hes3 transcription. In addition, there may be other target gene(s) of CXorf6 besides Hes3.
Several domains were identified in the CXorf6 protein in addition to the previously reported glutamineand proline-rich domains (1). In this regard, the evolutionally conserved MAML motif may play a critical role in the transactivating activity, because the L103P protein had a reduced transactivation function. The serine-rich domain may also be relevant to the transactivation (33). Furthermore, it is inferred that a nuclear localization signal resides on the N-terminal 123 amino acids and a nuclear body localization signal lies on amino acids 197-653, except for amino acids 567-607 encoded by exon 4. It might be possible that a nuclear body localization signal is preserved in the Q197X protein, and in the E124X protein as well, but could not function because of an aberrant protein folding.
Functional studies provided the molecular basis for the previously identified mutations and variations. The E124X and Q197X mutations previously have been shown to undergo NMD (3); if a small amount of truncated proteins were produced by mRNAs escaping NMD, such proteins would be nonfunctional. For the R653X mutation, although the artificially produced truncated protein had a normal transactivation function in vitro, the NMD analysis implies the occurrence of nearly complete NMD in vivo. The P286S, Q507R, and N589S proteins were confirmed to retain normal functions. These results are consistent with the previous genotype-phenotype correlations of the mutations and variations (3).
The siRNA experiments imply that CXorf6 is involved in testosterone production. In this context, it appears that testosterone production is not abolished in the absence of residual CXorf6 expression, because the degree of reduction was more obvious for the mRNA level than for the testosterone concentration. This would be consistent with the development of hypospadias in patients with CXorf6 nonsense mutations (3), because hypospadias is a phenotype caused by reduced, but not absent, testosterone effects around the critical period for sex development (4,25). When testosterone effects are abolished, FIGURE 5. The NMD analysis for the R653X mutation. A, the position of the R653X mutation and the examined regions. Region 1 spans exon 2 to exon 3 and encompasses the start codon. Region 2 spans exon 3 to exon 5 and involves the mutation. The gray regions represent the coding region, and the horizontal line denotes the untranslated regions. B, representative results of the patient. After 40 cycles of RT-PCR for region 1, no band is seen without CHX treatment (CHX(Ϫ)), and a clear band is observed after CHX treatment (CHX(ϩ)). GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene; N.C., negative control. C, representative results of the heterozygous mother. Left, X-inactivation analysis for a region encompassing the highly polymorphic CAG repeat tract and two methylation-sensitive HpaII sites at exon 1 of the androgen receptor gene. PCR products are obtained from both active and inactive X chromosomes before HpaII digestion and from inactive X chromosomes only after HpaII digestion. The comparison of the area under the curves between two heterozygous peaks (282 and 285 bp, marked with asterisks) before and after the HpaII digestion indicates that the two X chromosomes undergo random X-inactivation with a ratio of 40:60%. The small 276-and 279-bp peaks are byproducts of the slippage phenomenon. Right, RT-PCR direct sequencing for a region encompassing the mutation. The normal allele-only is delineated without CHX treatment (Ϫ), and the normal and nonsense alleles are identified with CHX treatment (ϩ). The sequencing of 100 RT-PCR clones revealed wild-type clonesonly (WT) without CHX treatment and both wild-type (n ϭ 56) and mutant (n ϭ 44) clones after CHX treatment. this results in female external genitalia in genetic males (25). Thus, it is likely that CXorf6 augments testosterone production.
The present study has implications for the molecular network involving CXorf6. HES3 was expressed in the human fetal testis, and the Hes3 promoter was transactivated by CXorf6. Sf1 is co-expressed with the mouse homolog for CXorf6 in fetal Sertoli cells and Leydig cells (3), and SF1 transactivated CXorf6. Thus, there may be an interaction among SF1, CXorf6, and HES3 in the fetal testis, which may play an important role in the testicular function including testosterone production. In this regard, although the mouse homolog for CXorf6 is not expressed in the adult testis, it is clearly expressed in the adult ovary (3) where SF1 (4,5) and HES3 are also expressed. In addition, premature ovarian failure has been described in a female with heterozygosity for a microdeletion involving CXorf6 (34). Thus, CXorf6 and its possible interaction with SF1 and HES3 may also be relevant to adult ovarian function.
In summary, the present study implies that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue for the elucidation of the CXorf6 function. On the basis of the characteristic structure, we have therefore designated CXorf6 as MAMLD1.