Browsing by Subject "Promoter"
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Item Epigenetic regulation of killer immunoglobulin-like receptor Gene expression in developing human natural killer cells.(2010-05) Cichocki, Frank M.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 therapiesItem Identification of novel signatures of murine definitive hematopoiesis(2014-01) Webber, BeauPluripotent stem cells (PSC) are a tantalizing prospect for a renewable source of patient-specific hematopoietic stem cells (HSC), however efforts to obtain PSC derived HSC capable of long-term engraftment have largely failed. We set out with the primary aim of identifying novel molecular signatures of definitive hematopoiesis, so that these signatures could be applied to improve generation and isolation of HSC in vitro. Toward this end we pursued both discovery and application based strategies centered on Runx1; a transcription factor that is critical for the development of definitive HSC. The discovery arm identified epigenetic modifications at Runx1 cis-regulatory elements that temporally associate with the transition from primitive to definitive hematopoiesis in vivo. We replicated these signatures in vitro by overexpressing HOXB4 in hematopoietic progenitors derived from murine embryonic stem cells (ESC), and found that HOXB4 directly interacts with the definitive-specific distal Runx1 promoter and mediates increased transcription, loss of DNA methylation, and acquisition of active histone modifications at this locus. We next applied our understanding of Runx1 regulation to generate a panel of clonal mESC lines harboring targeted, single-copy fluorescent reporters under the transcriptional control of Runx1 cis-regulatory elements. These lines were used to interrogate the hematopoietic activity of each element independent of copy number and chromosomal position, allowing us to identify combinations that provided optimal activity and fidelity. Building upon this, we established mESC lines harboring synthetic fluorescent and bioluminescent mini genes replicating the structure of the endogenous Runx1 locus and demonstrated that these lines reflect the dynamic promoter switching that occurs at Runx1 during hematogenesis. Sub-fractionation of embryoid body cells based on promoter activity revealed that nearly all colony forming cells (CFC) reside in the distal promoter expressing fraction. With this information we identified specific conditions that could further mature and expand distal positive cells. Collectively, this work identified a previously undescribed molecular signature of definitive hematopoiesis and the mechanism by which it is established. In addition, we applied this knowledge to generate tools with which to interrogate hematopoietic development in vitro, and have demonstrated their utility in optimizing strategies for obtaining definitive hematopoietic progenitors from PSC.