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dc.contributor.advisorGary S. Stein, Ph.D.
dc.contributor.authorDowdy, Christopher R.
dc.date2022-08-11T08:08:43.000
dc.date.accessioned2022-08-23T16:05:50Z
dc.date.available2022-08-23T16:05:50Z
dc.date.issued2012-05-25
dc.date.submitted2012-08-16
dc.identifier.doi10.13028/2mya-hk98
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31951
dc.description.abstractRunx1 is a master regulator of hematopoiesis, required for the initiation of definitive hematopoiesis in the embryo and essential for appropriate differentiation of many hematopoietic lineages in the adult. The roles of Runx1 in normal hematopoiesis are juxtaposed with the high frequency of Runx1 mutations and translocations in leukemia. Leukemia associated Runx1 mutations that retain DNA-binding ability have truncations or frame shifts that lose C-terminal domains. These domains are important for subnuclear localization of Runx1 and protein interactions with co-factors. The majority of leukemia associated Runx1 translocations also replace the C-terminus of Runx1 with chimeric fusion proteins. The common loss of Runx1 C-terminal domains in hematopoietic diseases suggests a possible common mechanism. We developed a panel of mutations to test the functions of these domains in vitro, and then developed mouse models to examine the consequences of losing Runx1 C-terminal domains on hematopoietic development and leukemogenesis in vivo. We previously observed that overexpression of a subnuclear targeting defective mutant of Runx1 in a myeloid progenitor cell line blocks differentiation. Gene expression analysis before differentiation was initiated revealed that the mutant Runx1 was already deregulating genes important for maturation. Furthermore, promoters of the suppressed genes were enriched for binding sites of known Runx1 co-factors, indicating a non-DNA-binding role for the mutant Runx1. To investigate the in vivo function of Runx1 C-terminal domains, we generated two knock-in mouse models; a C-terminal truncation, Runx1Q307X, and a point mutant in the subnuclear targeting domain, Runx1 HTY350-352AAA . Embryos homozygous for Runx1 Q307X phenocopy a complete Runx1 null and die in utero from central nervous system hemorrhage and lack of definitive hematopoiesis. Embryos homozygous for the point mutation Runx1HTY350-352AAA bypass embryonic lethality, but have hypomorphic Runx1 function. Runx1HTY350-352AAA results in defective growth control of hematopoietic progenitors, deregulation of B-lymphoid and myeloid lineages, as well as maturation delays in megakaryocytic and erythroid development. Runx1 localizes to subnuclear domains to scaffold regulatory machinery for control of gene expression. This work supports the role of transcription factors interacting with nuclear architecture for greater biological control, and shows how even subtle alterations in that ability could have profound effects on normal biological function and gene regulation.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectHematopoiesis
dc.subjectCore Binding Factor Alpha 2 Subunit
dc.subjectLeukemia
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectCell and Developmental Biology
dc.subjectCells
dc.subjectCirculatory and Respiratory Physiology
dc.subjectGenetic Phenomena
dc.subjectNeoplasms
dc.titleRunx1 C-terminal Domains During Hematopoietic Development and Leukemogenesis: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1607&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/604
dc.legacy.embargo2012-06-25T00:00:00-07:00
dc.identifier.contextkey3226741
refterms.dateFOA2022-08-24T02:54:37Z
html.description.abstract<p>Runx1 is a master regulator of hematopoiesis, required for the initiation of definitive hematopoiesis in the embryo and essential for appropriate differentiation of many hematopoietic lineages in the adult. The roles of Runx1 in normal hematopoiesis are juxtaposed with the high frequency of Runx1 mutations and translocations in leukemia. Leukemia associated Runx1 mutations that retain DNA-binding ability have truncations or frame shifts that lose C-terminal domains. These domains are important for subnuclear localization of Runx1 and protein interactions with co-factors. The majority of leukemia associated Runx1 translocations also replace the C-terminus of Runx1 with chimeric fusion proteins. The common loss of Runx1 C-terminal domains in hematopoietic diseases suggests a possible common mechanism. We developed a panel of mutations to test the functions of these domains <em>in vitro</em>, and then developed mouse models to examine the consequences of losing Runx1 C-terminal domains on hematopoietic development and leukemogenesis <em>in vivo</em>.</p> <p>We previously observed that overexpression of a subnuclear targeting defective mutant of Runx1 in a myeloid progenitor cell line blocks differentiation. Gene expression analysis before differentiation was initiated revealed that the mutant Runx1 was already deregulating genes important for maturation. Furthermore, promoters of the suppressed genes were enriched for binding sites of known Runx1 co-factors, indicating a non-DNA-binding role for the mutant Runx1.</p> <p>To investigate the in vivo function of Runx1 C-terminal domains, we generated two knock-in mouse models; a C-terminal truncation, Runx1<sup>Q307X</sup>, and a point mutant in the subnuclear targeting domain, Runx1 <sup> HTY350-352AAA </sup>. Embryos homozygous for Runx1 <sup> Q307X </sup> phenocopy a complete Runx1 null and die <em>in utero</em> from central nervous system hemorrhage and lack of definitive hematopoiesis. Embryos homozygous for the point mutation Runx1<sup>HTY350-352AAA</sup> bypass embryonic lethality, but have hypomorphic Runx1 function. Runx1<sup>HTY350-352AAA</sup> results in defective growth control of hematopoietic progenitors, deregulation of B-lymphoid and myeloid lineages, as well as maturation delays in megakaryocytic and erythroid development.</p> <p>Runx1 localizes to subnuclear domains to scaffold regulatory machinery for control of gene expression. This work supports the role of transcription factors interacting with nuclear architecture for greater biological control, and shows how even subtle alterations in that ability could have profound effects on normal biological function and gene regulation.</p>
dc.identifier.submissionpathgsbs_diss/604
dc.contributor.departmentRadiology
dc.description.thesisprogramCell Biology


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