Date of Degree

2-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Ben Ortiz

Committee Members

Laurel Eckhardt

Shubha Govind

Diana Bratu

Fei Li

Subject Categories

Biology | Biotechnology | Cell and Developmental Biology | Life Sciences

Keywords

Gene regulation, T cells, Insulators, Genome organization, Chromatin

Abstract

Genes of different spatiotemporal expression profiles are often juxtaposed in the genome. This organization raises risks of cross-regulatory influences from neighboring genes; for instance heterochromatin can spread over euchromatin or long-range acting enhancers can inappropriately activate genes. Gene regulatory elements such as Locus Control Regions (LCR) and Insulators prevent such cross-communications and allow for normal gene expression patterns. In transgenic systems, LCRs limit influences from surrounding chromatin by providing site-of-integration independent and specific spatiotemporal expression upon a linked transgene. The field’s understanding of the ability of an LCR to overcome chromatin influences and allow site-of-integration independent expression is minimal. Interestingly, this function of an LCR closely resembles that of barrier insulators. Barrier insulators prevent the spread of heterochromatin onto a euchromatin region and are characterized by their ability to suppress site-of-integration dependent chromatin influences upon a transgene. We hypothesize that the integration site-independence activity of LCRs is mediated by insulator-like DNA elements present within the LCR. In support of this hypothesis, we identify a novel barrier insulator activity within the mouse T-cell receptor (TCR)-a LCR. A 4.0-kb compilation of TCRa LCR sub-elements insulates a linked transgene in barrier assay- a long-term culture of stably transfected T cell lines. TCRa LCR-derived insulators enable maintenance of euchromatin and prevention of heterochromatin at a linked transgene. We find one element within the TCRa LCR that interacts with the USF1 transcription factor, which has been shown to have an important role in barrier insulation. In contrast to previously identified barrier insulators, the function of TCRa LCR-derived insulators does not require them to bi-laterally flank a gene. These data suggest that the TCRa LCR-derived elements may support both known and novel mechanisms of barrier insulation.

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