CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374290 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374290 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 228-273). Also available via World Wide Web.

Thesis (Ph.D.) -- McMaster University, 1999. / (Alpha) in title is a Greek letter. Includes bibliographical references (leaves 203-228). Also available via World Wide Web.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 140-161). Also available in electronic version.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.