In the past year significant progress has been made in the investigation of the function and transcriptional control of human class I major histocompatibility complex (MHC) receptors expressed on natural killer (NK) cells. We have identified and characterized a novel distal KIR promoter that is linked to the expression of KIR protein by mature NK cells, indicating that the primary role of the extensively studied KIR proximal promoter may be to act as a probabilistic transcriptional switch that controls the frequency of NK cells that express a given KIR allele. This discovery has important implications for the study of polymorphism that affect the intensity of KIR expression and susceptibility to disease. Our work has defined a novel paradigm for the selective activation of genes, and we are the pioneers in this area. Although the primary mechanism of stochastic decision-making by the KIR/Ly49 probabilistic switches is similar, the mechanism of KIR gene activation/silencing appears to be distinct from the Ly49 system. The KIR switch is positioned adjacent to the coding region, whereas the Ly49 switches are located further upstream. This arrangement leads to the production of double-stranded RNA (dsRNA) in the KIR proximal promoter region if the probabilistic switch is transcribing in the reverse direction, since the antisense transcripts overlap with distal forward KIR transcripts that are present throughout NK development. This discovery has important implications for the study of polymorphism that affect the intensity of KIR expression and susceptibility to disease. Our work has defined a novel paradigm for the selective activation of genes, and we are the pioneers in this area. Although the primary mechanism of stochastic decision-making by the KIR/Ly49 probabilistic switches is similar, the mechanism of KIR gene activation/silencing appears to be distinct from the Ly49 system. The KIR switch is positioned adjacent to the coding region, whereas the Ly49 switches are located further upstream. This arrangement leads to the production of double-stranded RNA (dsRNA) in the KIR proximal promoter region if the probabilistic switch is transcribing in the reverse direction, since the antisense transcripts overlap with distal forward KIR transcripts that are present throughout NK development. The possibility that siRNAs could be processed from the dsRNA prompted us to look for small RNAs derived from the KIR dsRNA region. We discovered a 28 base antisense RNA originating from the promoter region with properties similar to the piRNAs that have been associated with gene silencing in germ cells. In collaboration with Dr. Jeff Millers lab at the University of Minnesota, we have demonstrated a central role for the 28 base antisense RNA in KIR gene silencing, using the in vitro NK cell differentiation system developed by Dr. Miller. My group is also investigating polymorphisms in KIR promoter regions that affect either the frequency of NK cells expressing a given gene, or the level of KIR protein expression on the cell surface. Our current work is focussed on a polymorphism in the distal KIR promoter that inhibits distal transcription, resulting in greatly diminished KIR protein expression. Although we have previously demonstrated an important role for distal transcription in the activation of the proximal KIR promoter, this discovery reveals the importance of distal KIR transcript to interfere with KIR expression when over-expressed. Future plans include: investigation of the mechanism of action of the 28 base RNA; identifying and studying individuals with unusual patterns of KIR expression; analysis of KIR promoter polymorphisms and their association with the frequency of gene expression; designing reagents to control KIR gene activation for potential use in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010013-21
Application #
9343571
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Freund, Jacquelyn; May, Rebecca M; Yang, Enjun et al. (2016) Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells. PLoS Biol 14:e1002526
Li, H; Wright, P W; McCullen, M et al. (2016) Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes. Genes Immun 17:66-74
Felices, Martin; Lenvik, Todd R; Ankarlo, Dave E M et al. (2014) Functional NK cell repertoires are maintained through IL-2R? and Fas ligand. J Immunol 192:3889-97
Anderson, Stephen K (2014) Probabilistic bidirectional promoter switches: noncoding RNA takes control. Mol Ther Nucleic Acids 3:e191
Cichocki, Frank; Schlums, Heinrich; Li, Hongchuan et al. (2014) Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency. J Exp Med 211:1079-91
Wright, P W; Li, H; Huehn, A et al. (2014) Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression. Genes Immun 15:440-8
O'Connor, Geraldine M; Vivian, Julian P; Widjaja, Jacqueline M et al. (2014) Mutational and structural analysis of KIR3DL1 reveals a lineage-defining allotypic dimorphism that impacts both HLA and peptide sensitivity. J Immunol 192:2875-84
Cichocki, Frank; Miller, Jeffrey S; Anderson, Stephen K et al. (2013) Epigenetic regulation of NK cell differentiation and effector functions. Front Immunol 4:55
Wright, P W; Huehn, A; Cichocki, F et al. (2013) Identification of a KIR antisense lncRNA expressed by progenitor cells. Genes Immun 14:427-33
Barao, Isabel; Wright, Paul W; Sungur, Can M et al. (2013) Differential expression of the Ly49G(B6), but not the Ly49G(BALB), receptor isoform during natural killer cell reconstitution after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 19:1446-52

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