Regulation and Genetic Manipulation of Ligands for the Immunoreceptor NKG2D by Benjamin Gregory Gowen A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor David H. Raulet, Chair Professor Gregory M. Barton Professor Michael Rape Professor Karsten Gronert Spring 2015 Abstract Regulation and Genetic Manipulation of Ligands for the Immunoreceptor NKG2D by Benjamin Gregory Gowen Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor David H. Raulet, Chair NKG2D is an important activating receptor expressed by natural killer (NK) cells and some subsets of T cells. NKG2D recognizes a family of cell surface protein ligands that are typically not expressed by healthy cells, but become upregulated by cellular stress associated with transformation or infection. Engagement of NKG2D by its ligands displayed on a target cell membrane leads to NK cell activation, cytokine secretion, and lysis of the target cell. Despite the importance of NKG2D for controlling tumors, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. The work described in this dissertation was centered on the identification of novel regulators of ULBP1, one of the human NKG2D ligands. Using a forward genetic screen of a tumor-derived human cell line, we identified several novel factors supporting ULBP1 expression, and used the CRISPR/Cas9 system to further investigate these hits. Our results showed stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis, including transcription of the ULBP1 gene, splicing of the ULBP1 mRNA, and additional co-translational or post-translational regulation of the ULBP1 protein. One of the novel regulators of ULBP1 we identified was ATF4, a stress-induced transcription factor. ATF4 drives the constitutive expression of ULBP1 on some cancer cell lines and causes further upregulation of ULBP1 in response to stresses such as amino acid starvation or activation of the unfolded protein response. We found that ATF4 drives ULBP1 transcription by binding directly to the ULBP1 locus. The screen also identified the RNA-binding protein RBM4 as a driver of ULBP1 expression. Analysis of RBM4-deficient cells revealed that RBM4 suppresses a novel alternative splicing event in the ULBP1 transcript. The alternative isoform of ULBP1 mRNA is unlikely to encode a functional protein. As such, RBM4 supports ULBP1 expression by promoting the splicing of the functional mRNA isoform. This dissertation also describes the use of the CRISPR/Cas9 methodology to generate mice lacking two key NKG2D ligands, RAE-1ε and RAE-1δ. These mice are being used by other lab members to test hypotheses concerning regulation of NK cell activation imparted by RAE-1 expression on host cells within tumors. Additionally, these mice will soon be used to generate mice lacking all known NKG2D ligands. 1 The findings presented here highlight diverse mechanisms for the control of NKG2D ligand expression, and offer insight into the stress pathways that alert the immune system to danger. Furthermore, this work demonstrates the power of forward genetic screens and the CRISPR/Cas9 system for both discovery- and hypothesis-driven research. 2 Table of Contents Table of Contents ............................................................................................................................. i List of Figures and Tables............................................................................................................... ii Acknowledgements ........................................................................................................................ iv Chapter 1: Introduction ................................................................................................................1 Introduction to the immune system......................................................................................2 Natural killer cells ................................................................................................................3 NKG2D and its ligands ........................................................................................................4 Cell stress pathways and ATF4............................................................................................8 Chapter 2: Methodology..............................................................................................................12 Chapter 3: Identification of novel regulators of ULBP1 ..........................................................24 A genetic screen to identify regulators of human NKG2D ligand expression ..................25 Stepwise regulation of ULBP1 expression ........................................................................37 HSPA13 .............................................................................................................................37 SPCS1 and SPCS2 .............................................................................................................39 CRNKL1 ............................................................................................................................39 Genes involved in glycosylation ........................................................................................40 Functional assays ...............................................................................................................40 Failure of the ULBP2 screen..............................................................................................45 Discussion ..........................................................................................................................47 Chapter 4: Regulation of ULBP1 transcription by ATF4 ........................................................48 ATF4 supports ULBP1 expression on multiple tumor-derived cell lines ..........................49 ATF4 drives ULBP1 transcription in response to cell stress .............................................49 Additional treatments that may induce ULBP1 .................................................................63 Discussion ..........................................................................................................................67 Chapter 5: Regulation of ULBP1 mRNA splicing by RBM4 ...................................................69 Results ................................................................................................................................70 Discussion ..........................................................................................................................77 Chapter 6: Generation of Raet1 knockout mice ........................................................................79 Introduction ........................................................................................................................80 Generation of mutant mice, genotyping, and propagation of founders .............................81 Discussion ..........................................................................................................................86 Chapter 7: Conclusion .................................................................................................................87 References .....................................................................................................................................89 i List of Figures and Tables Figure 1-1: NKG2D ligands in humans and mice ...........................................................................5 Figure 2-1: Sequences of CRISPR/Cas9 target sites in HAP1 cells ..............................................15 Table 2-1: sgRNA sequences .........................................................................................................16 Table 2-2: PCR primers used to detect mutations .........................................................................17 Table 2-3: Primers used for qPCR .................................................................................................19 Table 2-4: Raet1 genotyping PCR primers ....................................................................................23 Figure 3-1: Expression of NKG2D ligands on HAP1 cells ...........................................................27 Figure 3-2: Genetic screens for drivers of ULBP1 and ULBP2 expression ..................................28 Table 3-1: List of genes enriched for gene-trap insertions in the ULBP1 screen ..........................30 Table 3-2: List of genes enriched for gene-trap insertions in the ULBP2 screen ..........................31 Figure 3-3: Target gene mutagenesis with the CRISPR/Cas9 system ...........................................33 Figure 3-4: Decreased ULBP1 expression upon loss of identified drivers ....................................35 Figure 3-5: Mutations specifically affect ULBP1 ..........................................................................36 Figure 3-6: Double and triple-mutant cell lines show stepwise decrease in ULBP1 expression ......................................................................................................38 Figure 3-7: Killing of HAP1 cells by activated mouse NK cells is primarily NKG2D-independent ..................................................................................................42 Figure 3-8: ATF4 KO HAP1 cells