Histone modifications are crucial in activities such as transcriptional activation, gene silencing, and epigenetic cellular memory. In particular, lysine acetylation via lysine (K) acetyltransferases (KATs) has been implicated in cancer development. Interestingly, KAT5, also known as Tip60 (tat-interactive protein-60kDa), has been reported to possess both tumor promoting and tumor suppressing properties depending on the context of malignancy. Herein we report that KAT5 contributes to tumorigenesis in epidermal growth factor receptor (EGFR) mutated lung cancer, and Kat5-knockout mice models demonstrate significantly reduced lung tumor burden. To probe the aberrant modification of KAT5, we demonstrated that KAT5 binds to and is phosphorylated by oncogenic EGFR in co-immunoprecipitation experiments. Next, to investigate whether KAT5 is involved in cell proliferation and survival, H1975 cells harboring L858R-T790M double-activating mutations were transfected with doxycycline inducible short helical RNA (shRNA) targeting KAT5 (shKAT5). Following treatment, shKAT5 cells were observed to have suppressed proliferation rates. Pharmacological inhibition using TH1834, a known KAT5 inhibitor, also suppressed proliferation rates in shKAT5 cells; in contrast BEAS2B cells, an immortalized normal human bronchial cell line, surprisingly exhibited increased viability compared to transformed human lung H1975 cells. This finding supports KAT5’s context-dependent role in in normal and abnormal cell homeostasis. To further investigate KAT5 in lung tumorigenesis in vivo, we generated EGFR-mutant conditional Kat5 knockout mice using a tetracycline-induced Cre/loxP system. Following doxycycline treatment for 10 weeks, isolated mice lungs for EGFRTL/CCSP-rtTA/Cre/Kat5F/F possessed significantly lower tumor volume compared to EGFRTL/CCSP-rtTA/Cre/Kat5wt/F and EGFRTL/CCSP-rtTA/Cre/Kat5wt/wt mice lungs. Hemotoxylin and eosin staining showed no evident hyperproliferation in lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5F/F mice whereas lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5wt/wt and EGFRTL/CCSP-rtTA/Cre/Kat5wt/F did, signifying that KAT5 has a potential regulatory role in cellular proliferation. RNA-Seq analysis of shKAT5 H1975 cells identified downstream targets involved in tumorigenic pathways. Subsequent quantitative polymerase chain reaction (PCR) of shKAT5 cells served to validate the reported targets. Taken together, these data offer insight into a KAT5 mediated oncogenic pathway that can provide novel therapeutic approaches in treating lung cancer.

Chromatin is a dynamic structure, and chromatin remodeling enzymes regulate chromatin structure to control gene expression and proper lineage specification. Tip60-p400 is a multi-subunit chromatin remodeling complex containing two biochemical activities: the Tip60 subunit is a lysine acetyltransferase (KAT) that targets histones and non-histone proteins, and p400 catalyzes ATP-dependent incorporation of histone variant H2AZ into chromatin. Both of these chromatin modifications have been widely studied with respect to gene expression, DNA damage repair, and apoptosis. Ablation of these catalytic subunits causes defects in normal embryonic development, ESC self-renewal, and gene expression. My goal has been to understand the multiple independent functions of Tip60-p400 acetyltransferase in ESC maintenance and embryonic development. I showed that Tip60 KAT function is dispensable for gene expression, chromatin accessibility, and ESC self-renewal, which is different from Tip60 knockdown phenotype. Interestingly, KAT deficient mutants exhibited defect in differentiation towards mesoderm and endoderm lineages. Consistent with this defect, I also observed gastrulation defect in mice lacking Tip60 KAT activity. Together, these data demonstrate that Tip60 KAT dependent function is only required during later stages of embryonic development, and it is dispensable for ESC self-renewal and pre-implantation development. Tip60 KAT contains four isoforms generated from alternative splicing, whose individual functions are poorly characterized. In the second part of this thesis, I investigated the developmental role of one of the isoforms of Tip60, called Tip55. Unlike Tip60 knockout mice, which lack all the isoforms and causes pre-implantation lethality, I found that ablation of Tip55 results in post-implantation lethality. I further found that loss of Tip55 causes defects in heart, and neural tube development, demonstrating the essential function of Tip55 isoform for organogenesis during embryonic development. Together, these studies have provided new insight into the functions of Tip60-p400 and the mechanisms by which this complex regulates gene expression, ESC pluripotency, and embryonic development. Furthermore, these studies set the stage for future work to identify how the catalytic and non-catalytic functions are directed to perform distinct regulatory functions, as well as how each Tip60 isoform individually contributes to formation of the mammalian body plan.