Yin Yang 1 (YY1) is a multifunctional transcription factor that is known mainly for its ability to activate or initiate transcription of a wide assortment of genes involved in cellular growth and differentiation. <i>Xenopus laevis </i>oocytes and embryos were used as a model to identify and characterize a potential developmental role for YY1. Northern and Western blots of oocyte and embryonic extracts showed YY1 mRNA and protein is expressed from the earliest stages of oocyte development through to tadpole stages. Examination of the transcriptional activity of YY1 in both oocytes and embryos using reporter gene constructs containing YY1-binding elements demonstrated that YY1 does not act as a repressor or activator of transcription either in oocytes or in embryos. Sub-cellular fractionation of oocytes and Western blot analysis showed YY1 is localized almost exclusively to the cytoplasm of oocytes and in cells of early embryos. Sequence analysis of YY1 revealed that it contains an established RNA binding motif located within the zinc fingers. A series of biochemical assays were performed to address the possibility that YY1 functions as a component of mRNPs in the oocyte cytoplasm. RNA gel mobility shift analyses using in vitro synthesized histone H2A transcripts and supershifts using YY1-specific antibodies suggested that YY1 or YY1-containing complexes in cytoplasmic extracts were able to bind RNA. Chromatographic analysis of oocyte lysates showed YY1 was specifically retained on oligo (dT) cellulose columns. Treatment of the same lysates with RNase abolished binding to oligo (dT), indicating that retention is dependent on the presence of intact polyadenylated RNAs. This suggested that YY1 may be a component of messenger ribonucleoprotein particles (mRNP). Separation of oocyte lysates by size exclusion chromatography (SEC) revealed that YY1 was present in large complexes with an approximate molecular mass of 480 kDa. RNase or phosphatase treatment of oocyte extracts released YY1 from high mass complexes. Analysis of phosphatase or RNase-treated extracts for DNA binding activity showed that monomeric YY1 was able to bind DNA with high affinity. Immunoprecipitation of YY1 complexes followed by cDNA synthesis and sequencing revealed that YY1 is associated with both ribosomal and messenger RNAs in the cytoplasm of the oocyte. These results indicate a novel function for YY1 as a component of messenger ribonucleoprotein particles.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-04102003-111738 |
| Date | 17 April 2003 |
| Creators | Ficzycz, Andrew Douglas |
| Contributors | Ovsenek, Nicholas (Nick), Juurlink, Bernhard H. J., Gloster, Andrew, Crawford, Michael, Bonham, Keith, Schreyer, David |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-04102003-111738/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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