Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1

Abstract

Epigenetic events refer to heritable changes in phenotypes that occur without alterations in the underlying DNA sequence. Among the major layers of epigenetic control, methylation of histone H3 at lysine 36 (H3K36) and lysine 27 (H3K27) defines euchromatin and facultative heterochromatin, respectively, thereby distinguishing transcriptionally active from repressive chromatin domains. These modifications are catalyzed by distinct families of histone methyltransferases: the Nuclear Receptor–Binding SET Domain Protein (NSD) family for H3K36me2 and the Polycomb Repressive Complex 2 (PRC2) for H3K27me3. NSD1, in particular, plays a crucial role in maintaining euchromatin integrity, and its loss or aberrant activation has been implicated in human congenital disorders, such as Weaver and Sotos syndromes, as well as broad types of cancers, including Diffuse Midline Glioma (DMG) and a subset of Acute Myeloid Leukemia (AML). This dissertation presents three studies focusing on the biochemical properties of NSD1, a large (~295 kDa) H3K36me2 methyltransferase essential for euchromatin regulation. Due to its size and extensive intrinsically disordered regions (IDRs), NSD1 has been difficult to purify and characterize. In the first study, I developed a baculovirus-insect cell system for expressing full-length (FL) NSD1 and SETD2. By isolating monoclonal baculovirus clones and optimizing a single-step FLAG purification protocol, I obtained highly pure recombinant proteins suitable for enzymatic assays. Importantly, both enzymes retained catalytic activities, representing the first successful reconstitution of full-length NSD1 and SETD2 in a defined biochemical system and enabling downstream structural and biochemical studies for the research community. In my second peer-reviewed publication, I investigated the molecular mechanisms that activate and regulate NSD1. Our study revealed that NSD1 requires allosteric activation through the aromatic pocket of its PWWP2 domain, which interacts directly with the nuclear paraspeckle protein NONO. This protein–protein interaction enhances the catalytic activity of NSD1 toward H3K36me2 deposition. Mouse embryonic stem cells harboring mutations within the PWWP2 aromatic pocket exhibit impaired differentiation into neural progenitor cells, a phenotype partially reproduced by NONO depletion. Intriguingly, NSD1 and NONO mutation are found to drive a rare and understudied macrocephaly phenotype in Sotos and MRXS34 syndromes, respectively. Together, these findings uncover a previously unrecognized mechanism of how nuclear paraspeckes regulate active chromatin, provide an insight into the molecular pathogenesis of macrocephaly, and highlight NSD1-PWWP2 as a vulnerability for therapeutic targeting of NSD1-dependent cancers. In my third peer-reviewed review article, I propose a model in which paraspeckles, NONO, and NSD1 cooperate to regulate euchromatin. We speculate on disease mechanisms driven by disruption of this axis and highlight future directions for targeting NSD1 in epigenetic therapy. Our findings reveal an unexpected layer of NSD1 regulation via paraspeckle-mediated allosteric control, with implications for chromatin state transitions during development and disease. In summary, this dissertation establishes a method for purifying full-length NSD1 and SETD2, overcoming longstanding technical challenges. It also identifies NONO as an allosteric activator of NSD1 and proposes a regulatory model linking paraspeckles to euchromatin dynamics. These findings advance our understanding of chromatin biology and provide a foundation for future therapeutic interventions for human pathological conditions.