The human Absent, small, or homeotic disc1 (ASH1L) is a member of the Trithorax group (TrxG) proteins that play a role in epigenetic gene activation of developmental HOX genes via H3K36me2 methylation mark. ASH1L contains the evolutionarily conserved SET domain responsible for catalyzing monomethylated and dimethylated lysine formation. The crystal structure of the SET domain of ASH1L revealed a substrate-binding pocket blockage caused by an autoinhibitory loop (AI-loop) that undergoes dynamic changes during catalysis and could be exploited for inhibitor development. Studies have shown that the AI-loop regulates the SET domain, thus the KMTase activity of ASH1L. The SET domain adopts an autoinhibited state where the AI-loop blocks the entry of substrate to the active site, have made it a difficult target for the development of inhibitors. The emerging ASH1L's role in multiple oncogenic processes leading to cancer makes it a viable therapeutic target. Effective targeted inhibition of ASH1L enzymatic activity would be a potential therapeutic approach in cancers driven by high HOX gene expression. We employed the state-of-the-art 1H and 13C-detected solution NMR to better understand the ASH1L regulatory mechanism. We investigated the AI-loop's dynamic structure and conformational mobility of backbone and side chains in the absence and presence of the first- in-class small molecule inhibitors. Numerous backbone amide signals across the AI loop and the catalytic cleft of the SET domain are being broadened, indicating the complex interplay of fast local to slow segmental dynamics across the ASH1L SET domain. The binding of the first-in-class inhibitors perturbs the signals around the AI-loop and SAM binding cleft, validating the inhibitor binding site in the solution. The recently published crystal structures of the MRG domain bound to the ASH1L SET domain revealed disordered conformations of the AI-loop and rearrangement in the SAM binding site compared to the apo ASH1L SET domain. It has been proposed that MRG15 allosterically activates ASH1L by releasing the AI loop. Therefore, we performed extensive studies in an aqueous solution to understand the role of MRG15 in stimulating the catalytic activity of ASH1L. We found that the full-length MRG15 is necessary to induce histone methyltransferase activity of the catalytic SET domain of ASH1L. In contrast, the MRG domain alone cannot enhance the catalytic activity. Furthermore, we found that only the complex of ASH1L SET domain with MRG15 but not with isolated MRG domain can interact with nucleosomes. In summary, I have established the direct link between the structural dynamics of the ASH1L SET domain and its enzymatic activity. Moreover, I have defined the adaptor role of the complete MRG15 protein as the substrate recognition factor for the ASH1L protein without perturbing the AI loop or SAM binding site. The atomic level studies mentioned above, supported by the detailed structure and dynamics studies of the first-in-class inhibitor complex with ASH1L, establish the solid foundations for further drug candidate development, selectively targeting the ASH1L and potentially other H3K36me2 methyltransferases.