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.

CTCF-independent, but not CTCF-dependent, elements significantly contribute to TCR-alpha locus control region activity.

The mouse TCRalpha/TCRdelta/Dad1 gene locus bears a locus control region (LCR) that drives high-level, position-independent, thymic transgene expression in chromatin. It achieves this through DNA sequences that enhance transcription and protect transgene expression from integration site-dependent position effects. The former activity maps to a classical enhancer region (Ealpha). In contrast, the elements supporting the latter capacity that suppresses position effects are incompletely understood. Such elements likely play important roles in their native locus and may resemble insulator/boundary sequences. Insulators support enhancer blocking and/or chromatin barrier activity. Most vertebrate enhancer-blocking insulators are dependent on the CTCF transcription factor and its cognate DNA binding site. However, studies have also revealed CTCF-independent enhancer blocking and barrier insulator activity in the vertebrate genome. The TCRalpha LCR contains a CTCF-dependent and multiple CTCF-independent enhancer-blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream elements located in the region of the LCR that supports CTCF-independent enhancer-blocking activity in cell culture. Internal deletion of either of these elements significantly impairs LCR activity. These results reveal that the position-effect suppression region of the TCRalpha LCR harbors an array of CTCF-independent, positive-acting gene regulatory elements, some of which share characteristics with barrier-type insulators. These elements may help manage the separate regulatory programs of the TCRalpha and Dad1 genes.