Abstract
The T cell rearrangement of the T cell receptor (TCR) genes TCRalpha and delta is specifically regulated by a complex interplay between enhancer elements and chromatin structure. The alpha enhancer is active in T cells and drives TCRalpha recombination in collaboration with a locus control region-like element located downstream of the Calpha gene on mouse chromosome 14. Twelve kb further down-stream lies another gene, Dad1, with a program of expression different from that of TCRalpha. The approximately 6-kb locus control region element lying between them contains multiple regulatory sites with a variety of roles in regulating the two genes. Previous evidence has indicated that among these there are widely distributed regions with enhancer blocking (insulating) activity. We have shown in this report that one of these sites, not previously examined, strongly binds the insulator protein CCTC-binding factor (CTCF) in vitro and in vivo and can function in an enhancer blocking assay. However, other regions within the 6-kb element that also can block enhancers clearly do not harbor CTCF sites and thus must reflect the presence of a previously undetected and distinct vertebrate insulator activity.
Highlights
The T cell receptor genes (TCR)1 play a central role in the development of T lymphocytes
Zhong and Krangel [16] suggested that the HS2– 6 is a boundary element that has an locus control region (LCR)-like activity, able to confer copy number-dependent and integration site-independent expression on an enhancer of the TCR␣ (E␣)containing transgene (4 –5) and able to protect both against ectopic activation of the TCR␣ by Dad1 regulatory elements and, reciprocally, against E␣ activation of Dad1 expression in T cells
Search for CCTC-binding factor (CTCF) Binding Sites within the TCR␣-Dad1 Region—Because the DNA region between the TCR␣ locus, which is only expressed in T cells, and the ubiquitous Dad1 gene (Fig. 1A) has been shown to possess an insulator activity [16], we searched for CTCF binding sites across the region flanked by the enhancer of the TCR␣ (E␣) and exon 3 of Dad1
Summary
Vol 279, No 24, Issue of June 11, pp. 25381–25389, 2004 Printed in U.S.A. Both CTCF-dependent and -independent Insulators Are Found between the Mouse T Cell Receptor ␣ and Dad Genes*. Zhong and Krangel [16] suggested that the HS2– 6 is a boundary element that has an LCR-like activity, able to confer copy number-dependent and integration site-independent expression on an E␣containing transgene (4 –5) and able to protect both against ectopic activation of the TCR␣ by Dad regulatory elements and, reciprocally, against E␣ activation of Dad expression in T cells They showed that HS2– 6 blocks an enhancer from activating a promoter when located between the two [16]. Our results suggest that the CTCF protein participates in the insulator activity of this region, allowing proper expression of TCR␣ and DadI genes, but is working in concert with previously unrecognized mechanisms of insulation
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