Date of Degree

2-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Benjamin D. Ortiz

Subject Categories

Allergy and Immunology | Biology | Immunology and Infectious Disease | Medical Immunology

Abstract

Orchestrated expression of multiple genes residing in the complex TCRα/δ/Dad1 locus requires tight control from multiple cis-acting elements. The TCRα locus control region (LCR), is positioned between TCRα and Dad1 gene, and has been implicated in the differential expression of both genes. In this study, we focus our work on the hypersensitive site (HS)1 prime (HS1'), located 3' of the classical Eα enhancer, within the TCRα LCR. We investigated its non- redundant role in TCRα expression in thymic and peripheral T cells as assayed by in vivo and in vitro studies. Furthermore, formation of HS1' in both lymphoid and non-lymphoid tissue raised the possibility of HS1' playing a dual role in regulating both the upstream (TCRα) as well as the downstream (Dad1) genes. To answer this question, we created wild type and mutant HS1' dual-reporter BACs utilizing human and rat CD2 reporter genes in the position of TCRα and Dad1, respectively. We find HS1' important for TCRα expression in thymus and spleen T cells, but dispensable for Dad1 expression. We widened our focus to include sequences outside of the TCRα LCR. Specifically, DNase I hypersensitivity assay revealed a cluster of active chromatin just 5' of the constant region Cα exons. Analysis of this 3.9-kb region using a BAC transgenic mouse model reveals its importance for TCRα gene expression in thymic and splenic T cells.Interestingly, this novel DNase hypersensitive regulatory complex will remain present upon the Vα-Jα rearrangement of the TCRα gene given its location 3' of the most downstream functional joining (J) segment, Jα2. Therefore, the novel cis-acting region may contribute to endogenous TCRα gene activity.

 
 

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