A new element within the T-cell receptor alpha locus required for tissue-specific locus control region activity

Abstract

Locus control regions (LCRs) are cis-acting regulatory elements thought to provide a tissue-specific open chromatin domain for genes to which they are linked. The gene for T-cell receptor alpha chain (TCRalpha) is exclusively expressed in T cells, and the chromatin at its locus displays differentially open configurations in expressing and nonexpressing tissues. Mouse TCRalpha exists in a complex locus containing three differentially regulated genes. We previously described an LCR in this locus that confers T-lineage-specific expression upon linked transgenes. The 3' portion of this LCR contains an unrestricted chromatin opening activity while the 5' portion contains elements restricting this activity to T cells. This tissue-specificity region contains four known DNase I hypersensitive sites, two located near transcriptional silencers, one at the TCRalpha enhancer, and another located 3' of the enhancer in a 1-kb region of unknown function. Analysis of this region using transgenic mice reveals that the silencer regions contribute negligibly to LCR activity. While the enhancer is required for complete LCR function, its removal has surprisingly little effect on chromatin structure or expression outside the thymus. Rather, the region 3' of the enhancer appears responsible for the tissue-differential chromatin configurations observed at the TCRalpha locus. This region, herein termed the "HS1' element," also increases lymphoid transgene expression while suppressing ectopic transgene activity. Thus, this previously undescribed element is an integral part of the TCRalphaLCR, which influences tissue-specific chromatin structure and gene expression.

FIG. 1

Diagram of the transgenic constructs used in this study. Four transgenic constructs are depicted with DNase I hypersensitive sites of the TCRα locus (as observed in T-cell lines) labeled with vertical arrows. Large arrows above the line indicate the predominant HS found in normal thymocytes. A horizontal arrow indicates the transcriptional orientation of the β-globin reporter gene, which is present in all four constructs. Tissue distribution and chromatin opening regions were described in reference .

FIG. 2

Copy number dependence of β:1-6 transgene transcription. (A) RNase protection assay with thymus RNA from four independent β:1-6 transgenic lines. Line numbers and their estimated relative copy number are indicated. Arrows indicate the signals from the human β-globin reporter transgene and endogenous TCRα signal. Migration of the full-length probes is shown in the probe lane. Non-Tg, nontransgenic. (B) PhosphorImager analysis of β:1-6 expression in thymus. RNase protection from experiment 1 is shown in panel A. Transgene expression was normalized to TCRα signal (defined as 1.0). Experiment 2 is a repeat of the previous experiment with different members of the same lines. Here, the transgene expression was normalized to the endogenous actin signal (see Materials and Methods), which is defined as 1.0 for this experiment. The ratio of transgene to endogenous control expression was then divided by the relative copy number to obtain the values presented. (C) Deletion of HS7 and -8 has a minor to negligible effect on transgene expression. PhosphorImager analysis of RNase protection assays. Thymus and spleen transgene expression was examined for three pairs of β:1-8 and β:1-6 transgenic mice. The relative β:1-8 and β:1-6 copy numbers are, respectively, as follows: pair 1, 2 and 16; pair 2, 21 and 17; and pair 3, 16 and 22. Transgene signal was normalized to endogenous actin signal (defined as 1.0) and divided by the copy number. Representative RNase protections are shown in Fig. 5A.

FIG. 3

β:1′-6 transgene expression is present in all founders, but is not as copy number dependent as the β:1-6 construct. (A) RNase protection assay with thymus RNA from eight independent β:1′-6 transgenic lines. Line numbers and their estimated relative copy numbers are indicated. Arrows indicate the signals from the human β-globin reporter transgene and endogenous TCRα signal (defined as 1.0). (B) PhosphorImager analysis of the experiment shown in panel A. Transgene signal was normalized to endogenous TCRα signal and then divided by the copy number.

FIG. 4

Tissue distribution of β:1-6 and β:1′-6 transgene expression. (A) RNase protection assay on RNA from the indicated tissues of β:1-6 line 41 (16 copies) and β:1′-6 line 13 (8 copies) transgenic mice. Arrows indicate signals from the β-globin and endogenous actin control. (B) PhosphorImager analysis of the experiment shown in panel A and an additional experiment using other β:1-6 (line 2, 17 copies) and β:1′-6 (line 47, 10 copies) transgenic lines (lower panel). Transgene mRNA signal was normalized to that of endogenous actin control (defined as 1.0) and then divided by the copy number.

FIG. 5

LCR HS deletion analysis. (A) RNase protection assays of the indicated organs of representative transgenic lines. The constructs are indicated. Relative copy numbers are as follows: β:1-8, 16; β:1-6, 22; β:1′-6, 43; and β:2-6, 28. Arrows indicate signals from human β-globin transgene mRNA and the endogenous actin control. (B) PhosphorImager analysis of RNase protection assays. Thymus and spleen transgene expression was examined for three pairs of β:1′-6 and β:2-6 transgenic mice. The relative β:1′-6 and β:2-6 copy numbers are, respectively, as follows: pair 1, 8 and 8; pair 2, 10 and 9; and pair 3, 43 and 28. Transgene signal was normalized to endogenous actin signal (defined as 1.0) and divided by the copy number. (C) PhosphorImager analysis of RNase protections performed with heart RNA from the same three pairs of transgenic mice used in panel B. Normalized transgene expression numbers were obtained as in panel B.

FIG. 6

Chromatin structure analysis at transgene loci. (Left panel) DNase I hypersensitivity assay of the indicated organs of β:1-6 line 41 transgenic mice. The parent band is a SwaI-SacI restriction fragment of the transgene. The probe is to the 5′ end of the fragment. The positions of HS clusters are indicated by brackets or arrows. (Right panel) DNase I hypersensitivity assay of the indicated organs of β:1′-6 line 14 transgenic mice. The parent fragment was generated as in panel A and detected with the same probe. Slopes indicate increasing DNase I concentration (general range, 0.0 to 4.0 μg/ml).

FIG. 7

Analysis of chromatin structure at the endogenous TCRαLCR. (A) DNase I hypersensitivity assay of the indicated organs from nontransgenic C57BL6/J mice. The parental band is an EcoRV restriction fragment of the TCRαLCR region. The probe is to the 3′ end of the fragment. HS clusters are indicated with brackets. (B) Similar assay with αβ thymocytes and γδ T cells isolated as described in Materials and Methods (99% purity). Slopes indicate increasing DNase I concentration. The middle lane (labeled BglII) contains EcoRV-BglII codigested genomic DNA. The BglII site is 15 nucleotides 5′ of the PmlI site and marks the junction of the HS1 and HS1′ clusters.