{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/140557"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/140557","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Hsu, Chen-I"],"institution":"Virginia Tech","degree_name":"Doctor of Philosophy","degree_level":"doctoral","degree_discipline":"Biomedical and Veterinary Sciences","degree_department":"Biomedical and Veterinary Sciences","school":null,"contributors":[],"advisors":[],"committee_chairs":["Yu, Jia-Ray"],"committee_members":["Mulvaney, Kathleen Michelle","Rocha, Pedro Fernandes","Xie, Hehuang David"],"year":2025,"date_issued":"2025-12-23","date_published":"2025-12-23","updated_at":"2026-07-22T22:19:16Z","subjects":["Epigenetic","Paraspeckles","H3K36me2"],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44968"],"render_values":[{"text":"vt_gsexam:44968","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/10919/140557","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Yu, Jia-Ray"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Mulvaney, Kathleen Michelle","Rocha, Pedro Fernandes","Xie, Hehuang David"]},{"key":"dc:contributor.department","label":"Department","values":["Biomedical and Veterinary Sciences"]},{"key":"dc:creator","label":"Author","values":["Hsu, Chen-I"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-12-24T09:00:35Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-12-24T09:00:35Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-12-23"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biomedical and Veterinary Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Epigenetic","Paraspeckles","H3K36me2"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44968"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/140557"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["DNA is the blueprint that carries all the genetic information of living organisms. In humans, every cell contains nearly identical copies of this DNA blueprint, yet cells become remarkably different, forming tissues such as muscle, skin, and brain. This diversity arises from a process called epigenetic regulation, which controls how genes are switched on or off without altering the DNA sequence itself. One way this occurs is through small chemical marks that attach to proteins called histones, which help organize DNA into compact structures known as chromatin. These marks determine which parts of the genome remain active and which stay silent. My dissertation focuses on a large enzyme called NSD1, which helps place one of these chemical marks to keep certain regions of DNA active. NSD1 is essential for normal growth and development, and when its activity is disrupted, it can lead to childhood overgrowth syndromes and several types of cancer. Because NSD1 is unusually large and flexible, it has been very challenging for scientists to purify and study. In the first part of my research, I developed a reliable method to produce the complete NSD1 protein in the laboratory. This method made it possible to examine its biochemical behavior in detail. In the second part, I discovered that NSD1 does not function alone. It must first be activated by another protein called NONO, which helps form small structures inside the nucleus known as paraspeckles. When NONO interacts with NSD1, it activates the enzyme, enabling it to place the necessary chemical marks on DNA-associated histones. Disrupting this interaction affects how stem cells develop into neurons, offering new clues about why certain genetic disorders impact brain development. The third part of my dissertation reviews recent research suggesting that NSD1 and paraspeckles work together to control how active regions of DNA are maintained and passed on when cells divide. This cooperation may also contribute to diseases caused by disruptions in chromatin regulation. Overall, this research establishes a framework for studying the full-length NSD1 protein, reveals how it is activated by NONO, and proposes a new model connecting paraspeckles to the regulation of active DNA. These findings deepen our understanding of how cells manage their genetic information and open potential directions for treating diseases related to errors in epigenetic control."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:title","label":"Title","values":["Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Yu, Jia-Ray"],"dc:contributor.committeemember":["Mulvaney, Kathleen Michelle","Rocha, Pedro Fernandes","Xie, Hehuang David"],"dc:contributor.department":["Biomedical and Veterinary Sciences"],"dc:creator":["Hsu, Chen-I"],"dc:date.accessioned":["2025-12-24T09:00:35Z"],"dc:date.available":["2025-12-24T09:00:35Z"],"dc:date.issued":["2025-12-23"],"dc:description.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."],"dc:description.abstractgeneral":["DNA is the blueprint that carries all the genetic information of living organisms. In humans, every cell contains nearly identical copies of this DNA blueprint, yet cells become remarkably different, forming tissues such as muscle, skin, and brain. This diversity arises from a process called epigenetic regulation, which controls how genes are switched on or off without altering the DNA sequence itself. One way this occurs is through small chemical marks that attach to proteins called histones, which help organize DNA into compact structures known as chromatin. These marks determine which parts of the genome remain active and which stay silent. My dissertation focuses on a large enzyme called NSD1, which helps place one of these chemical marks to keep certain regions of DNA active. NSD1 is essential for normal growth and development, and when its activity is disrupted, it can lead to childhood overgrowth syndromes and several types of cancer. Because NSD1 is unusually large and flexible, it has been very challenging for scientists to purify and study. In the first part of my research, I developed a reliable method to produce the complete NSD1 protein in the laboratory. This method made it possible to examine its biochemical behavior in detail. In the second part, I discovered that NSD1 does not function alone. It must first be activated by another protein called NONO, which helps form small structures inside the nucleus known as paraspeckles. When NONO interacts with NSD1, it activates the enzyme, enabling it to place the necessary chemical marks on DNA-associated histones. Disrupting this interaction affects how stem cells develop into neurons, offering new clues about why certain genetic disorders impact brain development. The third part of my dissertation reviews recent research suggesting that NSD1 and paraspeckles work together to control how active regions of DNA are maintained and passed on when cells divide. This cooperation may also contribute to diseases caused by disruptions in chromatin regulation. Overall, this research establishes a framework for studying the full-length NSD1 protein, reveals how it is activated by NONO, and proposes a new model connecting paraspeckles to the regulation of active DNA. These findings deepen our understanding of how cells manage their genetic information and open potential directions for treating diseases related to errors in epigenetic control."],"dc:description.degree":["Doctor of Philosophy"],"dc:format.medium":["ETD"],"dc:identifier.other":["vt_gsexam:44968"],"dc:identifier.uri":["https://hdl.handle.net/10919/140557"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Epigenetic","Paraspeckles","H3K36me2"],"dc:title":["Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Biomedical and Veterinary Sciences"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:16Z"}