Title

Unraveling the perforin gene in cytotoxic lymphocytes: The transcriptional territory of perforin and elements required for its locus control region

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Mathias G. Lichtenheld, Committee Chair

Abstract

Natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) directly recognize and eliminate virally infected and malignant host cells. The principal machinery they use in this capacity is the focused exocytosis of cytotoxic granules containing the lytic molecule perforin. The regulation of perforin expression during development and activation of NK cells and CTL is determined by transcription of the perforin gene. Nevertheless, 45kb of the human perforin locus does not contain the regulatory sequences sufficient to drive this process physiologically. Consequently, previous studies suggested that even more distal regulatory regions were required for normal perforin gene expression. To identify these potentially distal elements, we developed a new approach to conventional Deoxyribonuclease I hypersensitivity (DHS) assays that facilitates mapping DHS sites across 100kb regions in a single experiment. This method was combined with chromosome transfer to determine the long-range changes that occur to the chromatin structure of perforin and its neighboring genes during its de novo epigenetic and transcriptional activation. To that end, human chromosome 10 was transferred from fibroblasts, where perforin is epigenetically silent, to CTL where mouse perforin is expressed. The transchromosomal perforin gene was activated de novo and expressed at physiological levels in the CTL; this process was not co-regulated with its neighboring genes. Chromatin remodeling occurred across 150kb during epigenetic reprogramming and established a locus-wide domain over the perforin gene that was comprised of 15 DHS sites. We show with transgenes in the CTL model, and in lymphoid cells differentiated from transgenic mouse embryonic stem (ES) cells that this 150kb domain includes a locus control region (LCR), suggesting perforin was controlled physiologically. An engineered deletion in the transgenes and a spontaneous deletion in one transchromosomal perforin allele demonstrates that four distal regulatory sites are required for LCR function. These studies establish the complete network of cis-acting sequences responsible for transcriptional control of the cytolytic gene perforin. They are fundamental for continued dissection of the genetic and epigenetic mechanisms governing development and activation of the cytotoxic program in NK cells and CTL.

Keywords

Biology, Molecular; Health Sciences, Immunology

Link to Full Text

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