title: Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence.
abstract: 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.
authors.affiliation: Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan. mfukami@nch.go.jp
authors.forename: Maki
authors.initials: M
authors.lastname: Fukami
authors.forename: Yuka
authors.initials: Y
authors.lastname: Wada
authors.forename: Michiyo
authors.initials: M
authors.lastname: Okada
authors.forename: Fumiko
authors.initials: F
authors.lastname: Kato
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Katsumata
authors.forename: Takashi
authors.initials: T
authors.lastname: Baba
authors.forename: Ken-ichirou
authors.initials: K
authors.lastname: Morohashi
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Laporte
authors.forename: Motoo
authors.initials: M
authors.lastname: Kitagawa
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ogata
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007021:Hypospadias / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007984:Leydig Cell Tumor / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1 / Q000235:genetics / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1; D013739:Testosterone; D015534:Trans-Activators; D014157:Transcription Factors; C495457:HES3 protein, human; C411886:Hes3 protein, mouse; C471297:MAML2 protein, human; C528016:MAMLD1 protein, human; C515057:NR5A1 protein, human; C515058:steroidogenic factor 1, mouse