Regulatory mechanisms driving the random monoallelic expression of the natural killer cell receptor genes

AbstractRegulatory mechanisms driving the random monoallelic expression of the natural killer cell receptor genesbyDjem U. KissiovDoctor of Philosophy in Molecular and Cell BiologyUniversity of California, Berkeley,Professor David H. Raulet, Chair Natural killer (NK) cells constitute the first line of defense against many foreign pathogens and cancerous cells. Unlike T and B lymphocytes, NK cells do not rearrange their receptor genes and instead generate diversity for MHC I ligands by drawing on a pool of germline-encoded receptors in a stochastic fashion. These receptors are encoded by C-type lectin domain-containing genes in a tandem array on mouse chromosome 6, and are expressed in a random, monoallelic and mitotically stable pattern (RME). Genes are generally transcribed from both alleles, but in recent years RME has arisen as a notable exception and may describe up to ~10% of genes. While progress has been made toward the understanding of the prevalence and phenomenology of RME, the driving mechanisms are not understood. Research has been hampered by the lack of an established in vivo genetic model to dissect the role of specific regulatory elements in RME expression patterns. NK cell receptors provide the opportunity to generate such a model. NK cell receptors are regulated proximally, greatly simplifying the search for the relevant regulatory elements as they should occur near the gene locus itself. Furthermore, our lab has previously developed allele-specific antibodies, allowing the assessment of allelic expression on single cells rapidly and with a high degree of confidence by flow cytometry, circumventing the technical challenges, costs and time associated with experiments such as single cell RNA-seq. Finally, primary NK cells are readily cultured in vitro in medium containing IL-2 such that questions about mitotic stability of expression states are easily addressed. Deletion of enhancers in vivo has been greatly simplified with the advent of CRISPR/Cas9-based genome editing. Germline enhancer deletion in mice can now be achieved on timescales of months rather than years, and much more reliably than by traditional gene targeting methods. Additionally, analysis of the chromatin states of silent and active alleles has been historically limited by the requirement of large numbers of cells. Recent advances in chromatin profiling technologies (ATAC-seq and CUT&RUN) allow experiments to be performed with tens of thousands of cells rather than tens of millions, allowing the profiling of subsets of NK cells sorted with respect to allelic expression status using allele-specific antibodies. Using the power and flexibility provided by these new approaches, this thesis addresses the following questions. First, what is the role of enhancer elements in regulating the expression frequencies of the variegated NK receptor genes? Chapter 3 addresses this question through a series of enhancer deletions and F1 hybrid genetics in vivo. Furthermore, in Chapter 3 I leverage the power of allele-specific antibodies and flow cytometry to search for RME expression patterns in genes previously thought to be ubiquitously expressed by a given cell type. Strikingly, RME-like expression patterns are identified in all assayed receptors: NKG2D by NK cells, CD45 by T cells and B cells, CD8 by cytotoxic T cells, and Thy1 by both cytotoxic and helper T cells. This supports a model where RME is the consequence of generalized stochastic properties of gene expression and can be detected in many and perhaps all genes. Next, this thesis addresses the chromatin features of both silent and active NK receptor gene alleles in vivo and deduces clues as to the mechanism of mitotic stability in RME. Chapter 4 discusses the results of chromatin analyses in sorted primary cells from F1 hybrid mice. Additionally, this chapter addresses the role of enhancer elements in the maintenance of active RME alleles. The data presented in this thesis results in a unified model of RME and enhancer function derived from the broadly probabilistic properties of gene expression. Enhancers display constitutive activation but only probabilistic effects on target gene expression, suggesting enhancer activation is generally decoupled from target gene activation. Deletion of individual enhancers from a set of enhancers regulating a target gene results in a reduction of the proportion of cells expressing the gene at both the Ly49g and Nkg2d loci. A particularly notable result is the transformation of the (apparently) ubiquitously expressed Nkg2d gene to a stable RME gene via enhancer deletion, displaying all the fundamental properties of the natural RME NK receptor genes. These deletions had no large effect on the expression level of these genes per cell. These results strongly support the binary on/off model of enhancer action. Rather than being specific to a set of genes, stochastic allele activation appears to be a general property of gene expression and is not restricted to a biologically meaningful set of genes. Surprisingly, silent NK receptor alleles lack a repressive chromatin state, and more closely resemble the chromatin of lineage non-specific genes. Mitotic stability in RME is likely a result of allelic classification as lineage appropriate or lineage non-specific by stochastic enhancer action. We propose that previously documented examples of RME are extreme manifestations of a general property, rather than a result of a dedicated mechanism. RME, therefore, does not seem to be an exception to the rules and instead describes gene expression broadly. This model of gene expression conceptually resembles a bistable multivibrator, in which an initial signal determines one of two possible states which are then maintained in the absence of the initial signal. In the context of developmental gene regulation, this signal is presumably provided during cellular differentiation, and may be provided to inducible genes in fully differentiated cells. Importantly, gene induction (whether developmental or through some stimulatory signal) is read out by the varying strength of enhancer activity as a rheostat, raising or lowering the probability of stable allelic activation

Differential Role of Hematopoietic and Nonhematopoietic Cell Types in the Regulation of NK Cell Tolerance and Responsiveness.

Many NK cells express inhibitory receptors that bind self-MHC class I (MHC I) molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self-MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self-tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with wild-type (WT) and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient nonhematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus nonhematopoietic cells may be imposed by distinct mechanisms

Differential Role of Hematopoietic and Nonhematopoietic Cell Types in the Regulation of NK Cell Tolerance and Responsiveness

Many NK cells express inhibitory receptors that bind self MHC class I molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with WT and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I-deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient non-hematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus non-hematopoietic cells may be imposed by distinct mechanisms

Binary outcomes of enhancer activity underlie stable random monoallelic expression

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum