Date

2014

Author

Güpür, Gizem

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17β-estradiol (E2), the main circulating estrogen in the body, is involved in physiological regulation of many tissue and organ functions, including mammary tissue. E2 is also involved in target tissue malignancies. E2 regulates cellular proliferation, differentiation and death in target tissues. The lasting effects of E2 on cells are mediated by estrogen receptor and β that are the products of distinct genes and act as transcription factors. Upon binding to E2, the activated ER regulates the expression of E2 target genes through ERE (estrogen response element)-dependent and ERE-independent signaling pathways. The ERE-dependent signaling pathway refers to transcription events initiated by the interaction of E2-ER with ERE sequences. The transcription regulation involving the functional interactions of E2-ER with other transcription factors bound to their cognate response elements on DNA is called as the ERE-independent signaling pathway. In a microarray study conducted in our laboratory to identify genes involved in ERE-dependent and ERE-independent signaling pathways, YPEL2, a member of the highly conserved Yippee-like (YPEL) gene family, is suggested to be an E2 responsive gene regulated through the ERE-dependent signaling pathway. The YPEL gene family, named after Drosophila Yippee protein, has 100 members which share an extremely high amino-acid sequence identity in 68 species ranging from yeast, C.elegans, flies, plants to mammals. The members of the human YPEL genes, YPEL1-5, encode putative zinc binding small proteins with molecular vi masses ranging from 13,500 to 17,500 Da. Although structures and functions of Ypel proteins are yet unclear, a limited number of studies suggests the involvement of Ypel proteins in development, cell cycle progression and mitosis, as well as cellular senescence and death. Our analyses using various bioinformatics tools suggest that Ypel proteins share a high degree of structural and functional properties that might be important for basic cellular processes. Our bioinformatics analyses also suggest that each YPEL gene is spatiotemporally regulated by different repertoire of transcription factors which may be activated by distinct signaling pathway in response to different internal and external clues. To analyze the synthesis and intracellular localization of Ypel2, we initially cloned cDNAs of all five members of the human YPEL family, using a cDNA library from ER-positive MCF7 cell line derived from a breast adenocarcinoma, for comparisons. We then showed that the un-liganded ER regulates basal mRNA levels of YPEL2. Moreover, the expression of YPEL2, as well as YPEL3, is repressed by E2. These findings are consistent with our prediction that YPEL2 and YPEL3 are E2 and ER responsive genes. We found that Ypel1, 2 and/or 3 are synthesized in COS7, derived from transformed African green monkey kidney fibroblast-like cells, and localized to a region just outside of the nucleus, however we could not detect any endogenous Ypel protein in MCF7 cells. On the other hand, we observed that over-expressions of YPEL1-5 lead to the leakage of DNA from the nucleus into the cytoplasm in a pattern that overlaps with the localization of each Ypel protein in COS7 and MCF7 cells, in the latter the over-expression of Ypel1-5 is associated with a gross deterioration of the nuclear lamina integrity. Future studies will address the regulation of YPEL2 expression as well as the functions of Ypel2 in cell models.

Subject Keywords

Estrogen., Estrogen, Molecular cloning., Proteins.

URI

http://etd.lib.metu.edu.tr/upload/12617667/index.pdf
https://hdl.handle.net/11511/23845

Collections

Graduate School of Natural and Applied Sciences, Thesis