Charles E. Whitehurst

Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8α specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.

Control of V(D)J Recombinational Accessibility of the Dβ1 Gene Segment at the TCRβ Locus by a Germline Promoter

Abstract The germline promoter region upstream of the Dβ1 gene segment in the murine TCRβ locus was deleted to assess its role in controlling V(D)J recombination. Associated with diminished Dβ1 region germline transcription, rearrangement of the Dβ1 but not the Dβ2 gene segment was reduced 10- to 20-fold. A corresponding reduction in RAG-mediated cleavage at the Dβ1 and Jβ1 signal sequences was apparent only when purified CD4 − CD8 − thymocytes were analyzed because, as we demonstrate, cleavage at these gene segments also occurred in CD4 + CD8 + thymocytes. These findings suggest that germline promoters regulate localized accessibility of gene segments for recombination and that in CD4 + CD8 + thymocytes TCRβ allelic exclusion does not result from inaccessibility of Dβ gene segments.

Deletion of Germline Promoter PDβ1 from the TCRβ Locus Causes Hypermethylation that Impairs Dβ1 Recombination by Multiple Mechanisms

Abstract The role of the germline transcriptional promoter, PDβ1, in V(D)J recombination at the T cell receptor β locus was investigated. Deletion of PDβ1 caused reduced germline transcription and DNA hypermethylation in the Dβ1-Jβ1 region and decreased Dβ1 rearrangement. Analyses of methylation levels surrounding recombination signal sequences (RSS) before, during, and after recombination revealed that under physiological conditions cleavage of hypomethylated alleles was preferred over hypermethylated alleles. Methylation of a specific CpG site within the heptamer of the 3′ Dβ1 RSS was incompatible with cleavage by the V(D)J recombinase. These findings suggest that methylation can regulate V(D)J recombination both at a general level by influencing regional chromatin accessibility and specifically by blocking RSS recognition or cleavage by the V(D)J recombinase.

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

We have previously identified a DNase I-hypersensitive site in the T cell receptor β locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Eβ 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 MARβ. 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 MARβ represses Eβ-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARβ from the endogenous locus does not change T cell receptor β 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.

The T-Cell Receptor β Variable Gene Promoter Is Required for Efficient Vβ Rearrangement but Not Allelic Exclusion

ABSTRACT To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the Vβ13 promoter was either deleted (P13−/−) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13R/R). In P13−/− mice, cleavage, rearrangement, and transcription of Vβ13, but not the flanking Vβ gene segments, were significantly inhibited. In P13R/R mice, inhibition of Vβ13 rearrangement was less severe and was not associated with any apparent reduction in Vβ13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal Vβ13-recombination signal sequence junction and Vβ13 coding joint formation of both wild-type and mutant Vβ13 alleles. However, a low level of aberrant Vβ13 cleavage was consistently detected, especially in TCR transgenic P13R/R mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated Vβ gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant Vβ cleavages during allelic exclusion.

The T cell receptor β enhancer promotes access and pairing of Dβ and Jβ gene segments during V(D)J recombination

The precise function of cis elements in regulating V(D)J recombination is still controversial. Here, we determined the effect of inactivation of the TCRβ enhancer (Eβ) on cleavage and rearrangement of Dβ1, Dβ2, Jβ1, and Jβ2 gene segments in CD4-CD8- [double-negative (DN)] and CD4+CD8+ [double-positive (DP)] thymocytes. In Eβ-deficient mice, (i) Dβ1 rearrangements were more severely impaired than Dβ2 rearrangements; (ii) most of the Dβ and Jβ cleavages and rearrangements occurred in DP, rather than in DN, thymocytes; and (iii) most of the 3′ Dβ1 cleavages were coupled to 5′ Dβ2 cleavages instead of to Jβ cleavages, resulting in nonstandard Dβ1-Dβ2-Jβ2 joints. These findings suggest that the Eβ regulates TCRβ rearrangement by promoting accessibility of Dβ and Jβ gene segments in DN thymocytes and proper pairing between Dβ1 and Jβ gene segments for cleavage and joining in DP thymocytes.

Normal TCRβ transcription and recombination in the absence of the Jβ2–Cβ2 intronic cis element

Abstract The developmental regulation of antigen receptor gene transcription and recombination are mediated by cis regulatory elements. At the T cell receptor β chain locus ( TCRβ ), two DNase I hypersensitive sites within the Jβ2–Cβ2 intron contained binding sites for NF-κB and additional nuclear factors and were postulated to be involved in controlling TCRβ transcription and V(D)J recombination. To test this possibility, we deleted these elements from the mouse genome by homologous recombination and assayed the effect on transcription of both the germline and rearranged TCRβ locus, and on TCRβ rearrangement in T and B lymphocytes. We found that TCRβ transcription and V(D)J recombination and T cell development were normal in these mutant mice. Therefore, the Jβ2–Cβ2 intronic elements are dispensable for TCRβ assembly and function.