The 3'-Jα Region of the TCRα Locus Bears Gene Regulatory Activity in Thymic and Peripheral T Cells

Abstract

Much progress has been made in understanding the important cis-mediated controls on mouse TCRα gene function, including identification of the Eα enhancer and TCRα locus control region (LCR). Nevertheless, previous data have suggested that other cis-regulatory elements may reside in the locus outside of the Eα/LCR. Based on prior findings, we hypothesized the existence of gene regulatory elements in a 3.9-kb region 5' of the Cα exons. Using DNase hypersensitivity assays and TCRα BAC reporter transgenes in mice, we detected gene regulatory activity within this 3.9-kb region. This region is active in both thymic and peripheral T cells, and selectively affects upstream, but not downstream, gene expression. Together, these data indicate the existence of a novel cis-acting regulatory complex that contributes to TCRα transgene expression in vivo. The active chromatin sites we discovered within this region would remain in the locus after TCRα gene rearrangement, and thus may contribute to endogenous TCRα gene activity, particularly in peripheral T cells, where the Eα element has been found to be inactive.

Fig 1. DNase I hypersensitivity sites (HS) within the Jα2 to Cα1 region.

(A) Spleen T cell nuclei from C57BL/6N mouse were subjected to DNase I titration. Arrows indicate the positions of the 6.7-kb NdeI parent fragment and HS. Results from two independent, representative experiments are shown. (B) Scaled diagram of the approximate locations of detected HS (arrows). The thick arrow near Jα1 indicates the most prominent HS.

Fig 2. Diagram (not to scale) of the TCRα/Dad1 dual-reporter BAC construct [14].

Horizontal arrows indicate the orientation of the two reporter genes. A Vα17 promoter drives expression of a genomic human CD2 reporter gene (ghCD2). The rat CD2 reporter is driven by the Dad 1 promoter. Vertical arrows indicate the location of the HS of the TCRα LCR (including the TCRα enhancer, Eα). The 3.9-kb region deletion in the mutant BAC is marked and runs from 38-bp 5’ of the SacI site through 30-bp 5’ of the end of Cα exon 1.

Fig 3. Impaired hCD2 reporter expression in the absence of the deleted region.

Flow cytometry analyses of human CD2 (hCD2) and rat CD2 (rCD2) reporter gene expression in spleen T cells (TCRβ⁺) from the indicated, independent wild type (Wt) and mutant (Mt) dual-reporter BAC transgenic mouse lines. Reporter gene expression in transgenic (solid line) and non-transgenic control (dashed line) cells is shown.

Fig 4. Absence of the deleted region impairs upstream, but not downstream reporter gene expression in thymocytes.

(A) Northern blot analyses of human and rat CD2 reporter gene mRNA levels in thymocytes from the indicated lines of wild type and mutant reporter BAC transgenic mice. 18S rRNA signals are used as a loading control. Relative transgene copy number for each mouse line is indicated. The black line indicates excision of samples from the blot that are irrelevant to the present study. Panels B and C depict PhosphorImager analyses of the human CD2 (B) and rat CD2 (C) reporter mRNA signals detected by northern blots. The normalized mRNA levels (per transgene copy) from each wild type (black bars) and mutant (white bars) transgenic mouse line are graphed relative to each other (as % maximum). (D) Statistical analyses of the above data using the two-tailed students t test. Graph bars indicate the average (+/- S.E.) normalized mRNA levels among the lines. The asterisk indicates the statistical significance of the difference in hCD2 mRNA levels between wild type and mutant BAC (p = 0.012). In contrast, no significant difference in rCD2 mRNA levels was detected (p = 0.942).

Fig 5. The deleted region is functional in peripheral T cells.

qRT-PCR analyses of human (A) and rat (B) CD2 reporter gene mRNA levels (+/- S.E.) in isolated spleen T cells (SpT) from the indicated lines of wild type (black bars) and mutant (white bars) transgenic mice. Observed reporter mRNA levels per copy from each transgenic line are graphed relative to each other (as % maximum). hCD2 reporter mRNA levels were normalized to endogenous TCRα mRNA levels, and rCD2 expression were normalized to endogenous Dad1 mRNA levels using primers that detect sequences not present in the reporter BAC. Three experiments (S1 Table) were performed in duplicates. (C) Statistical analyses of the above data using the two-tailed students t test. The asterisk indicates the statistical significance of the difference in hCD2 mRNA levels between wild type and mutant BAC (p = 0.016). In contrast, no significant difference in rCD2 mRNA levels was detected (p = 0.819). Graph bars indicate the average (+/- S.E.) normalized mRNA levels among the lines.

Fig 6. Signatures of active chromatin in the 3’ Jα region.

Visualization of histone H3 acetylation marks in CD4⁺,CD8⁺ thymocytes (top track) and activated peripheral CD4⁺ T lymphocytes (bottom track) assayed by ChIP-seq. Shown are screenshots of tracks obtained from publicly available data via the NCBI Epigenomics Browser. The region depicted spans the mouse TCRα gene locus DNA (chromosome 14) containing the Jα2 and Jα1 segments, and the Cα1 constant region exon. Top row: (Unpublished data). ChIP-seq in Mus musculus, strain 129SvJae x C57BL/6 (H3K9ac2). Accession number: ESX000004775. Bottom row: [22]. ChIP-seq in Mus musculus, strain C57BL/6 (H3K9K14ac). Accession number: ESX000001399.