Functional domains of Runx1 are differentially required for CD4 repression, TCRbeta expression, and CD4/8 double-negative to CD4/8 double-positive transition in thymocyte development.
Runx1 (AML1) has multiple functions in thymocyte development, including CD4 repression in immature thymocytes, expression of TCRbeta, and efficient beta-selection. To determine the functional domains of Runx1 important for thymocyte development, we cultured Runx1-deficient murine fetal liver (FL) cells on OP9-Delta-like 1 murine stromal cells, which express Delta-like 1 and support thymocyte development in vitro, and introduced Runx1 or C-terminal-deletion mutants of Runx1 into the FL cells by retrovirus infection. In this system, Runx1-deficient FL cells failed to follow normal thymocyte development, whereas the introduction of Runx1 into the cells was sufficient to produce thymocyte development that was indistinguishable from that in wild-type FL cells. In contrast, Runx1 mutants that lacked the activation domain necessary for initiating gene transcription did not fully restore thymocyte differentiation, in that it neither repressed CD4 expression nor promoted the CD4/8 double-negative to CD4/8 double-positive transition. Although the C-terminal VWRPY motif-deficient mutant of Runx1, which cannot interact with the transcriptional corepressor Transducin-like enhancer of split (TLE), promoted the double-negative to double-positive transition, it did not efficiently repress CD4 expression. These results suggest that the activation domain is essential for Runx1 to establish thymocyte development and that Runx1 has both TLE-dependent and TLE-independent functions in thymocyte development.
Kawazu
Masahito
M
Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Asai
Takashi
T
Ichikawa
Motoshi
M
Yamamoto
Go
G
Saito
Toshiki
T
Goyama
Susumu
S
Mitani
Kinuko
K
Miyazono
Kohei
K
Chiba
Shigeru
S
Ogawa
Seishi
S
Kurokawa
Mineo
M
Hirai
Hisamaru
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000378:metabolism*; D016827:CD8 Antigens / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D050676:Core Binding Factor Alpha 2 Subunit; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005333:Fetus / Q000166:cytology; D022781:Hepatocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D011247:Pregnancy; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
D015704:CD4 Antigens; D016827:CD8 Antigens; D050676:Core Binding Factor Alpha 2 Subunit; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; C493726:Runx1 protein, mouse; D014157:Transcription Factors