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Gene Enhancer1
* Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Científicas (CSIC) Parque Tecnológico de Ciencias de la Salud, Armilla (Granada), Spain; and
Department of Immunology, Duke University Medical Center, Durham, NC 27710
During thymocyte maturation, enhancers of genes encoding for TCR
(Tcrd) and TCR
(Tcra), E8, and E, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an E fragment, T1-T2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire E or T1-T4. These experiments indicated that T3-T4 collaborates with factors bound to T1-T2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on T1-T2 explained the molecular basis for requirement of intact T2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of E, EMC, in which E myb and runx sites have been substituted for T2 runx and ets sites, that argues against the notion of a requirement for strict E enhanceosome structure for function. EMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an E enhanceosome are rather flexible. Activation of V(D)J recombination by EMC during thymocyte development resulted, however, to be premature and indistinguishable from that of T1-T2. These results indicate that T3-T4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between T2 runx/ets sites binding proteins and proteins bound to T3-T4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on E might represent a more flexible form of information processing during thymocyte development.
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1 This work was supported by the Spanish Ministry of Science and Innovation Grant BFU2008-01651 and a grant from the Fundación Mútua Madrileña de Investigación Médica.
2 B.d.B. and J.L.R. contributed equally to this work.
3 Current address: Department of Pediatrics, Division of Allergy/Immunology, Duke University Medical Center, Durham, NC 27710.
4 Current address: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
5 N.Z. and N.B.-D. contributed equally to this work.
6 Current address: Department of Biology, University of Konstanz, Konstanz, Germany.
7 Address correspondence and reprint requests to Dr. Cristina Hernández-Munain, Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Científicas, CSIC, Parque Tecnológico de Ciencias de la Salud, Armilla, (Granada), Spain. E-mail address: chmunain{at}ipb.csic.es
8 Abbreviations used in this paper: DN, double negative; DP, double negative; E, Tcra locus enhancer; E, Tcrd locus enhancer; ATF, activation transcription factor; TCF-1, T cell factor-1; LEF-1, lymphocyte enhancer-binding factor-1; ChIP, chromatin immunoprecipitation; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element binding protein; CBP, CREB-binding protein; DMS, dimethylsulfate; HAT, histone acetyltransferase; HDAC, histone deacetylase; R, RAG-1–/–; Rxβ, RAG-2–/– x TCRβ; RT, room temperature; HEB, HeLa E-box binding protein.
9 The online version of this article contains supplementary material.
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