A Nuclear Matrix Attachment Region Upstream of the T Cell Receptor β Gene Enhancer Binds Cux/CDP and SATB1 and Modulates Enhancer-dependent Reporter Gene Expression but Not Endogenous Gene Expression

Samit Chattopadhyay,Charles E Whitehurst,Jianzhu Chen

doi:10.1074/jbc.273.45.29838

Samit Chattopadhyay, Charles E Whitehurst + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.45.29838

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Abstract

We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.

Highlights

The rearrangement and expression of the T cell receptor ␤ (TCR␤)1 gene is essential to early T lymphocyte development [1]
To identify protein factors that bind to HS1, we performed Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts from DP thymocytes because HS1 was most prominent in these cells [37]
Despite the presence of the consensus nuclear factor binding sites within probe III, it did not generate any complex in EMSA (Fig. 1B, lanes 5 and 6)

Summary

The abbreviations used are

TCR␤, T cell receptor ␤; E␤, TCR␤ gene enhancer; IgH, immunoglobulin heavy chain; Ig␬, immunoglobulin ␬ light chain; MAR, nuclear matrix attachment region; HS, DNase I-hypersensitive sites; EMSA, electrophoretic mobility shift assay; DN, CD4ϪCD8Ϫ double negative; DP, CD4ϩCD8ϩ double positive; bp, base pair(s); kb, kilobase pair(s); DTT, dithiothreitol. The TCR␤ gene enhancer (E␤) is the only cis-regulatory element demonstrated to be required for both the lineage- and stage-specific transcription and rearrangement of the TCR␤ gene [7,8,9,10,11,12]. MARs are typically AT-rich DNA sequences that bind to the nuclear matrix, often contain topoisomerase II cleavage sites, and exhibit a propensity for base unpairing when subjected to superhelical strain [21, 22] They have been proposed to be involved in transcription, DNA recombination, replication, and repair [23]. To characterize novel cis-regulatory elements involved in controlling TCR␤ gene rearrangement and/or transcription, we previously screened a 100-kb region of the TCR␤ locus and identified along with E␤ 10 additional DNase I-hypersensitive sites (HS) [37]. We localize nuclear factor binding sites within HS1, characterize two nuclear factors that bind to HS1, demonstrate that HS1 is a nuclear matrix attachment region, and reveal the potential pitfalls of functional analyses of MARs outside of their natural chromosomal context

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION