Browsing by Author "He, YouWen"

Myeloid/lymphoid or mixed-lineage leukemia (MLL)-family genes encode histone lysine methyltransferases that play important roles in epigenetic regulation of gene transcription, and these genes are frequently mutated in human cancers. While MLL1 and MLL4 have been the most extensively studied, MLL2 and its homolog MLL3 are not well-understood. Specifically, little is known regarding the extent of global MLL2 involvement in the regulation of gene expression and the mechanism underlying its alterations in mediating tumorigenesis. To study the role of MLL2 in tumorigenesis, we somatically knocked out MLL2 in a colorectal carcinoma cell line, HCT116. We observed that the MLL2 loss of function results in significant reduction of cell growth and multinuclear morphology. We further profiled MLL2 regulated genes and pathways by analyzing gene expression in MLL2 wild-type versus MLL2-null isogenic cell lines. Our results reveal the connection of MLL2 to multiple cellular signaling pathways and suggest potential mechanisms underlying tumorigenesis mediated by MLL2 alterations.

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

To maintain homeostasis, T lymphocytes die through caspase–dependent apoptosis. However, blockage of caspase activity in T lymphocytes does not increase cell survival. The loss of caspase 8 activity leads to programmed necrosis (necroptosis) upon T cell receptor (TCR) stimulation in T lymphocytes. Necroptosis is correlated with excessive macroautophagy, an intracellular catabolic process characterized by the sequestration of cytoplasmic compartments through double–membrane vacuoles. Meanwhile, the proper induction of macroautophagy is required for T lymphocyte survival and function. Cellular caspase 8 (FLICE)–like inhibitory protein (c–FLIP) promotes survival in T lymphocytes. c–FLIP suppresses death receptor–induced apoptosis by modulating caspase 8 activation. Whether this modulation plays a role in the regulation of necroptosis has yet to be studied. Additionally, overexpression of c–FLIP reduces autophagy induction and promotes cell survival in cell lines. It remains unclear whether c–FLIP protects primary T lymphocytes by regulating the threshold at which autophagy occurs. In this study, c–FLIP isoform–specific conditional deletion models were used to study the role of c–FLIP in necroptosis and autophagy in primary T lymphocytes.

Our results showed that the long isoform of c–FLIP (c– FLIP_L) regulates necroptosis by inhibiting receptor interacting protein 1 (RIP–1). Upon TCR stimulation, c–FLIP_L–deficient T cells underwent RIP–1–dependent necroptosis. Interestingly, though previous studies have generally described necroptosis in the absence of caspase 8 activity and apoptosis, pro–apoptotic caspase 8 activity and the rate of apoptosis were also increased in c–FLIPL–deficient T lymphocytes. Moreover, c– FLIP_L–deficient T cells exhibited enhanced autophagy, which served a cytoprotective function.

Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway), or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies in c–FLIP–deficient T lymphocytes suggested that c–FLIP promotes cell survival in the absence of death receptor signals. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of caspases. Whether c–FLIP regulates mitochondrion–dependent apoptotic signaling remains unknown. Here, by deleting the c–Flip gene in mature T lymphocytes, we showed a role for c–FLIP in the intrinsic apoptosis pathway. In naïve T cells stimulated with the apoptosis inducer, c–FLIP suppressed cytochrome c release from mitochondria. Bim–deletion rescued the enhanced apoptosis in c–FLIP–deficient T cells, while inhibition of caspase 8 did not. Different from activated T cells, there were no signs of necroptosis in c–FLIP–deficient naïve T cells. Together, our findings indicate that c–FLIP is a key regulator of apoptosis, necroptosis and autophagy in T lymphocytes.