No description available.

Retinoic acid (RA) is critical for embryonic development and cell differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in Pre-â cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents them from differentiating to either endodermal or neuronal cells. This suggests that PBX is an important regulator of RA-induced differentiation of P19 cells. A microarray analysis was performed to identify PBX regulated genes, utilizing the empty vector P19 (TO3) and antisense to PBX (AS2) cell lines, during RA-induced differentiation of P19 cells into endodermal or neuronal cells. Among the genes identified by the microarray, Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor 1 (SF-1) were identified to be directly or indirectly regulated by PBX. Both DAX-1 and SF-1 proteins have only recently been reported to be present in preimplantation mouse embryos prior to the expression of steroidogenic enzymes, suggesting they may play a role in early mouse embryogenesis. To determine the roles of DAX-1 and SF-1 during RA-dependent differentiation, P19 cells that inducibly express either FLAG-DAX-1 or FLAG-SF-1 upon removal of doxicyclin were prepared. We found that overexpression of FLAG-DAX-1 had no effect on the RA-induced differentiation of P19 cells. However, FLAG-SF-1 overexpression prevented the RA-dependent loss of Oct-4, DAX-1 and the increase in COUP-TFI, COUP-TFII, and Ets-1 mRNA levels during the commitment stages of both endodermal and neuronal differentiation. Surprisingly, continued expression of SF-1 for seven days caused a RA-independent loss of Oct-4 protein. However, cells which continued to express SF-1 for seven days did not terminally differentiate into endodermal or neuronal cells in response to RA treatment. In addition, we found evidence for a feedback loop, where PBX reduces SF-1 mRNA expression and continued SF-1 expression blocks the RA-dependent increase in PBX protein levels. Our findings suggest that SF-1 plays a novel role in P19 cells where its level of expression is critical for the differentiation state of the cells. At basal levels SF-1 maintains the pluripotent state of the cells, while SF-1 levels must be dramatically reduced for cells to differentiate into both endodermal and neuronal cells upon RA treatment. However, at elevated levels above basal, SF-1 inhibits Oct-4 expression and leads to the induction of the expression of steroidogenic enzymes with a pattern consistent with adrenal cells in a RA-independent fashion. Taken together these data suggest that SF-1 plays a much more dynamic role in P19 cells than previously reported. / Biochemistry

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

The C4 cycle represents a series of biochemical and anatomical modifications that are targeted to overcome the effects of photorespiration caused by the oxygenase capability of Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO). The cycle has evolved independently in over 60 lineages, which suggests that recruitment of genes into the C4 cycle is a relatively easy process. However, the mechanisms by which the anatomy and cell-specificity of the components of the C4 cycle is achieved is poorly understood. Preliminary work in maize indicated several components of the C4 cycle may be targeted by microRNAs (miRNAs). To explore this, a library of sRNA sequences from mature leaf tissue of the model C4 species Cleome gynandra L. was generated and then searched against a list of expressed sequence tag sequences for candidate genes of the C4 cycle. To complement this, transgenic C. gynandra containing the viral p19 protein, which is capable of suppressing miRNA activity, were produced. A limited subset of the candidate C4 genes showed a high level of sRNA read alignment. In C. gynandra plants expressing p19 photosynthesis was compromised and transcripts of several genes (most notably RbcS and RCA) were upregulated. These data were complemented by examining the effect of illumination on developing C. gynandra cotyledons, and attempts to generate a hybrid between C. gynandra and the C3 C. hassleriana Chodat. RbcS also showed elevated abundance in etiolated cotyledons, although this rapidly declined after illumination. The remainder of the C4 genes profiled accumulated in etiolated tissue, but were upregulated within 6 hours of illumination. Therefore, this study has illustrated that miRNA activity may play a role in maintaining the C4 photosynthetic cycle at optimum efficiency, although it has not been possible to identify at which point(s) this regulation is applied. Secondly, RbcS appears to be subject to multiple regulatory mechanisms in C. gynandra, and it is possible that miRNAs have a role in negatively regulating expression of RbcS.

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

No description available.