Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1.
Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5' of the B cell-specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Danae
D
Schulz
Lothar
L
Vassen
Kwan T
KT
Chow
Sarah M
SM
McWhirter
Rupesh H
RH
Amin
Tarik
T
Möröy
Mark S
MS
Schlissel
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D042002:Chromatin Assembly and Disassembly; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015723:Gene Library; D018390:Gene Targeting; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; C079873:Gfi1 protein, mouse; C486395:Gfi1b protein, mouse; D018398:Homeodomain Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C064658:RAG-1 protein