Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5′-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes

Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element

HLA-B7 reporter mRNA expression in B cells is not suppressed by the presence of a second reporter gene upstream of the TCRα LCR.

<p>PhosphorImager analyses of representative northern blot (bottom right inset) analyzing levels of spleen B cell expression of hCD2 and HLA-B7 mRNA relative to those observed in thymus (designated as 100% for all lines) in three independent lines (10, 17, 22) of hCD2:1-8:B7 transgenic mice. Reporter signals are normalized to 18S loading control signal. HLA-B7 expression levels in B cells (B), relative to thymus (T), from this dual-reporter transgene construct is similar to that seen in single-reporter 1-8:B7 transgenic mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015527#pone-0015527-g005" target="_blank">Fig. 5</a>). Levels of hCD2 mRNA from the dual-reporter hCD2:1-8:B7 transgene are as low, relative to thymus, as the hCD2 protein signals detected in flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015527#pone-0015527-g004" target="_blank">Fig. 4</a>).</p

Lymphoid organs express the highest levels of both hCD2 and HLA-B7 reporter transgenes.

<p>PhosphorImager analyses of northern blots of RNA prepared from the indicated tissues of the hCD2:1-8 (<b>A</b>), hCD2:1-8:B7 (<b>B</b>) or 1:8-B7 (<b>C</b>) transgenic mice. Reporter mRNA levels are quantified and normalized to 18S rRNA signal. Y-axis values represent the mean (+/− S.D.) expression levels relative to the thymus (designated as 100%) observed among three independent lines of mice bearing the indicated transgene.</p

The genomic locus of the TCRα LCR and transgene constructs.

<p>(<b>A</b>) Scale diagram of the TCRα/Dad1 genomic locus containing the TCRα LCR. (<b>B</b>) Diagrams (not drawn to scale) of the three heterologous TCRα LCR reporter transgenes used in these studies. Note that the hCD2 transgene is in the position of the TCRα gene with respect to the LCR sequences. In contrast, the HLA-B7 reporter gene is in the position of the Dad1 gene. Vertical arrows and numbers indicate the nine identified DNase I hypersensitive sites (HS) of the LCR. Horizontal arrows indicate the transcription orientation of the genes depicted. Solid boxes indicate exons. The asterisk denotes the placement of a premature stop codon.</p

The TCRα LCR drives significant mRNA expression levels of a 3′-linked HLA-B7 reporter gene in B cells.

<p>(<b>A</b>) Northern blot analyses of RNA prepared from thymocytes and isolated spleen B cells from three independent lines of 1:8-B7 transgenic mice. Thy  =  Thymus, SpB  =  Spleen B cells, NTG  =  non-transgenic. (<b>B</b>) Graph depicting PhosporImager analyses of HLA-B7 reporter expression levels normalized to 18S rRNA. Y-axis values are expressed relative to thymus mRNA levels (designated as 100%).</p

Ectopic TCRα/hCD2 reporter gene activity is absent in B cells of TCRα/Dad1 dual-reporter gene BAC transgenic mice.

<p>(<b>A</b>) Real-time reverse transcriptase-mediated PCR experiments on RNA prepared from thymocytes and isolated B cells from individual TCRα/Dad1 dual-reporter BAC transgenic mice. Y-axis values indicate the relative hCD2-Cα reporter (open bars) and endogenous TCRα (filled bars) mRNA levels within an individual mouse (thymocyte level designated as 100%). hCD2-Cα reporter mRNA signals ranged from ∼10–20% of endogenous TCRα mRNA levels. The normalizing control mRNA was β-actin. Experiments on two separate individual mice per each of two independent BAC transgenic lines are shown. (<b>B</b>) The Dad1 promoter on the BAC is active in B cells. Real time RT-PCR detection of BAC-resident, Dad1 promoter-driven rCD2 reporter expression from B cells isolated from individual BAC transgenic mice. The data are normalized to endogenous Dad1 mRNA levels.</p

Increased T cell-selectivity of epigenetic hCD2 promoter chromatin activation in the dual-reporter transgene context.

<p>(<b>A</b>) Chromatin immunoprecipitation assay on thymocytes and isolated spleen B cells detecting the trimethyl-lysine 4 epigenetic mark on Histone H3 from single reporter (hCD2:1-8 line 4) and dual-reporter (hCD2:1-8:B7 line 10) transgenic mice. Y-axis values represent the ratio of percent H3K4me3 marks obtained at the hCD2 promoter to that detected at the endogenous GAPDH promoter, an internal standard used here as a normalizing control. (<b>B</b>) Confirmation of ChIP results using distinct, independent lines of transgenic mice bearing single reporter (hCD2:1-8 line 29) and dual reporter (hCD2:1-8:B7 line 22) transgenes. For both experiments, the Y-axis values are derived from the formula hCD2 [H3K4me3 – IgG/input]/GAPDH [H3K4me3 – IgG/input].</p