Critical requirement of GABPalpha for normal T cell development.
GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Ralpha expression, GABP is critically required for TCR rearrangements and hence normal T cell development.
Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Shuyang
S
Yu
Dong-Mei
DM
Zhao
Raja
R
Jothi
Hai-Hui
HH
Xue
D000818:Animals; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D005434:Flow Cytometry; D051268:GA-Binding Protein Transcription Factor / Q000037:antagonists & inhibitors / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D015321:Gene Rearrangement; D053263:Gene Regulatory Networks; D019426:Integrases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000378:metabolism; D020395:Receptors, Interleukin-7 / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D051268:GA-Binding Protein Transcription Factor; C495656:Gabpa protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016693:Receptors, Antigen, T-Cell, alpha-beta; D020395:Receptors, Interleukin-7; D014157:Transcription Factors; C415350:interleukin-7 receptor, alpha chain; C045073:Cre recombinase; D019426:Integrases