Project Abstract Epigenetic modifications at transcriptional regulatory regions play an important role in facilitating lineage-specific gene expression during stem cell differentiation. In addition to lineage-specific transcription factors, Trithroax-group (TxG)-group proteins promote lineage-specific gene expression through antagonizing the Polycomb-mediated transcriptional repression. In mammals, Mll1/Mll2 (Mixed Lineage Leukemia) and Ash1L (Absent, Small, or Homeotic discs 1-Like) complexes are two TxG complexes that mediate covalent histone modifications through their histone methyltransferase (HMTase) activities towards histone H3 lysine 4 and histone H3 lysine 36 respectively. Although previous genetic studies have revealed that Mll1/Mll2 and Ash1L complexes are functionally involved in a common epigenetic regulatory process, it remains unknown how these two complexes are connected at the molecular level to carry out their functions in transcriptional activation. Additionally, in contrast to the well-studied Mll1/Mll2 complexes and histone H3K4 methylation, the functions of Ash1L and its mediated histone H3K36 methylation at promoters are largely unknown. In an effort to address these fundamental questions, we purified the Ash1L-interacting proteins and identified Spindlin1 (Spin1), a histone H3K4me3-specific reader, physically binds to Ash1L. Deletion of either Ash1L or Spin1 in mouse embryonic stem cells impairs the expression of early lineage-specific genes upon induced differentiation, suggesting a functional connection between Spin1 and Ash1L in cells. Built upon these results, we propose a new model to unify the function of Mll1/Mll2 and Ash1L in transcriptional activation. In this model, Mll1/Mll2, Spin1, and Ash1L form an epigenetic regulatory axis, in which the individual component is sequentially recruited to the lineage-specific gene promoters to mediate histone modifications during transcriptional activation. Specifically, Spin1 plays a central role in connecting Mll1/Mll2 and Ash1L by recruiting Ash1L to the H3K4me3-marked lineage-specific gene promoters. To understand the functional role of individual components in this Mll1/Mll2-Spin1-Ash1L epigenetic regulatory axis, we will combine biochemical assays, CRISPR/Cas9- mediated genome editing, and next generation sequencing-based genetic analysis to dissect individual regulatory step proposed in the working model. Specifically, we will determine (1) whether Ash1L and its HMTase activity are required for its function in facilitating transcriptional activation; (2) the molecular mechanisms for the recruitment of Ash1L to gene promoters; (3) the structural basis underlying the interaction between Ash1L, Spin1 and histone H3K4me3. Completion of this study will not only significantly advance our understanding on the basic epigenetic mechanisms regulating the lineage-specific gene activation, but also reveal new therapeutic targets for blocking aberrant gene activation in cancers.