Differentially expressed nucleolar TGF-beta1 target (DENTT) in mouse development.
Differentially expressed nucleolar TGF-beta1 target (DENTT) is a recently identified gene whose mRNA is differentially affected by TGF-beta1 in TGF-beta1-responsive human lung cancer cells and who is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) protein superfamily. Here, we report that mouse DENTT mRNA contains a 2031-bp open reading frame that encodes a predicted polypeptide of 677-amino acids with a relative molecular mass of 77,671 Da. The mouse and human DENTT sequences show 77% and 78% homology at the nucleotide and amino acid level, respectively. Mouse DENTT is predicted to be a nuclear protein with two nuclear localization signals (NLS), two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. Green fluorescent protein (GFP)-tagged full-length mouse DENTT transfected into COS-7 cells showed localization predominantly in the nucleolus. Reverse transcription-polymerase chain reaction amplification, Northern hybridization, and Western blot analyses showed expression of mouse DENTT mRNA and protein throughout mouse embryogenesis. Immunohistochemical staining analysis showed that DENTT is expressed in multiple tissues in a defined spatiotemporal pattern during mouse embryogenesis. The heart and primitive brain were the first organs of the embryo that showed immunoreactivity for the DENTT antibody by day 8 of development (E8). In the developing mouse brain, the choroid plexus was intensely stained for DENTT in all stages of development. The spinal cord and dorsal root ganglia were also positive for DENTT staining beginning in the 11-day-old embryo (E11), where homogeneous immunostaining was observed throughout the developing neurons. By day 16 of development (E16), only a small subset of the neuronal population in the spinal cord and dorsal root ganglia was positively stained for DENTT. DENTT immunoreactivity increased steadily with maturation as the differentiation of cartilage and osteoblasts proceeded and reached a maximum in the growth plate during endochondral ossification. DENTT expression was also detected in multiple rodent cell types in vitro, including mouse F9 embryonal carcinoma (EC) cells. Addition of retinoic acid or sodium butyrate to F9 EC cells showed a rapid decrease in expression of DENTT protein occurring by 1 hr that continued to decrease to almost undetectable levels after 24 hr. Cotransfection of full-length mouse DENTT expression plasmid with 3TPLux or COL7A1Luc Luciferase reporter plasmids into F9 EC cells significantly increased the level of 3TPLux reporter transcription while decreasing the level of COL7A1Luc reporter transcription, suggesting that DENTT may play multiple roles in modulating transcriptional responses. These findings suggest new roles for the TTSN superfamily during embryogenesis and differentiation.
Ozbun
Laurent L
LL
National Cancer Institute, Cell and Cancer Biology Branch, Rockville, Maryland 20850, USA.
Martínez
Alfredo
A
Angdisen
Jerry
J
Umphress
Sarah
S
Kang
Yang
Y
Wang
Min
M
You
Ming
M
Jakowlew
Sonia B
SB
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001842:Bone and Bones / Q000196:embryology; D002319:Cardiovascular System / Q000196:embryology; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000502:physiology; D002466:Cell Nucleolus / Q000502:physiology*; D002823:Chorion / Q000737:chemistry; D004268:DNA-Binding Proteins; D004064:Digestive System / Q000196:embryology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D006321:Heart / Q000196:embryology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009420:Nervous System / Q000196:embryology; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C486649:DXBwg1396e protein, mouse; D009687:Nuclear Proteins; D012333:RNA, Messenger; C508960:TGFB1 protein, human; C432963:TSPYL2 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1