The immune system is our primary defense against infection and disease.
Immune cells need to recognize and efficiently destroy invasive pathogens while, at the
same time, exercising tolerance towards normal cells and tissues within the body.
Because pathogenic organisms are constantly evolving to evade detection, the immune
system must employ multiple recognition strategies to keep pace. Natural killer (NK)
cells have evolved a self versus non-self recognition strategy known as “missing self”
that is based upon the recognition of self major histocompatibility complex (MHC)
molecules by stochastically expressed inhibitory receptors on the surface of NK cells.
When MHC expression is downregulated by a virus or cellular transformation event, the
dampening signals that balance against NK cell activation are lost due to a lack of
inhibitory receptor engagement. This lack of inhibitory signaling, along with the
engagement of activating receptors, leads to the elimination of the distressed cell
through targeted NK cell-mediated cytotoxicity. The work presented in this manuscript
focuses on the transcriptional regulation of a critically important family of human NK cell
inhibitory receptors known as killer immunoglobulin-like receptors (KIR).
The KIR genes are present within the leukocyte receptor complex on
chromosome 19 and are expressed in a variegated, clonally restricted pattern on fully
differentiated NK cells. How this pattern of gene expression is regulated during NK cell
development is not well understood despite the demonstrated clinical relevance of KIR
during hematopoietic cell transplantation to treat patients with leukemia, the influence of
the KIR repertoire on the progression of HIV to AIDS, and the importance of KIR during
pregnancy. Progress in the elucidation of how KIR genes are regulated has been slow due to the complexity of the KIR locus and the lack of KIR genes in mice, which are
much more amenable to genetic manipulation.
We have shown that the 5’ upstream regulatory region of each KIR gene
contains a previously uncharacterized distal promoter with a functional c-Myc binding
site. Stimulation of primary peripheral blood NK cells with IL-15 induces c-Myc binding at
the distal promoter, which acts to promote KIR transcription. We also found that the
overexpression of c-Myc protein in the NK92 cell line, which lacks surface KIR due to
dense methylation of CpG dinucleotides proximal to the transcriptional start site, causes de novo surface KIR expression. Taken together, these results suggest that IL-15
directly promotes KIR transcription by inducing the binding of c-Myc to the distal
promoter. We hypothesize that the recruitment of c-Myc and the initiation of active
transcription from the distal promoter may also be key steps in the removal of repressive
epigenetic marks within KIR promoters during human NK cell development to allow for
stable gene expression.
In addition to identifying a novel distal promoter, our group has found that the
conventional proximal KIR promoter exhibits bi-directional transcriptional activity,
meaning that transcription can initiate in either the sense or antisense orientation. We
observed a strong inverse correlation between the expression of KIR antisense
transcripts and receptor expression on the cell surface, leading to the hypothesis that
antisense transcripts directly participate in RNA-mediated transcriptional repression of
individual KIR genes. We found that over-expressing full-length antisense transcripts during NK cell development led to an approximately 70% reduction in KIR expression
compared to controls. Furthermore, we determined that full-length antisense transcripts
are processed into a 28 base RNA with biochemical properties similar to those attributed
to members of the PIWI family of small RNAs. We also demonstrate that the 28 base sequence is necessary for antisense transcript-mediated repression of KIR gene
expression. This work establishes a direct association between KIR antisense
transcription and the initiation of DNA methylation within the KIR promoter. Further
elucidation of the mechanisms that regulate KIR expression during NK cell development
may provide a basis for new strategies in the design of NK cell-based therapies
University of Minnesota Ph.D. dissertation. May 2010. Major: Microbiology, Immunology and Cancer Biology. Advisor: Jeffrey S. Miller, M.D. 1 computer file (PDF); ix 93 pages.
Cichocki, Frank M..
Epigenetic regulation of killer immunoglobulin-like receptor Gene expression in developing human natural killer cells..
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