Personnel
Barbara Knowles
Co-Director, IMB
Senior Staff Scientist
The Jackson Laboratory
(207) 288-6361
barbara.knowles@jax.org
http://www.jax.org/staff/barbara_knowles
Laboratory members:
Mimi de Vries, Research Scientist
Alexei Evsikov, Associate Research Scientist
Anne Peaston, Associate Research Scientist
Karen Fancher, Graduate Student
Transitions: Systems approaches to the oocyte-to-embryo transition and the stem cell-to-malignant precursor cell transition in mammary tumorigenesis
Oocyte-to-embryo transition: My laboratory explores the molecular mechanisms driving reprogramming of the egg and sperm nuclei between fertilization and activation of the newly formed, combined embryonic genome.
Oocytes transcribe mRNAs during their growth in the ovarian follicle and when fully grown become transcriptionally inert. Timely translation of these early maternal messages provides one mechanism for molecular change in this transcriptionally silent cell. Stabilization of these maternal mRNAs is accomplished by specific proteins, which bind to cis-elements in their 3'-untranslated regions, preventing them from being polyadenylated and translated. Combined computational and molecular approaches to identify common cis-elements (3'UTRs) of these mRNAs forms one arm of our research. Dr. Mimi de Vries is collaborating with Dr. Joel Graber, to examine the 3'UTRs of specific maternal messages for common cis-element motifs.
Dr. Alexei Evsikov found a series of known and suspected RNA-binding proteins are very abundantly expressed in the fully grown oocyte. These proteins protect the maternal mRNAs from degradation by binding to cis-elements in their 3’UTR. When these bound proteins are modified, by signal transduction mechanisms, as the result of hormonal stimulation, the mRNAs can be polyadenylated and translated. Dr. Evsikov also identified an oocyte-specific form of the translation factor, Eif4E, which plays a central role in the initiation of translation. Interplay between these proteins serves to initiate translation of specific proteins providing the changing biochemical environment necessary for the full grown oocyte to complete maturation, meiosis, fertilization, and formation of a totipotent embryonic blastomere.
Drs. Evsikov and Anne Peaston found that MaLR retroviral transcripts are transcribed in the growing oocyte and are extremely abundant during the oocyte–to-embryo transition. Furthermore, when these LTRs integrate into host genes they coordinate host gene expression, in some cases upregulating transcription of normal host genes, in some cases providing de novo oocyte expression and in some cases providing new start sites for transcription resulting in truncated proteins. Interestingly these retroviral transcripts, from both the sense and anti-sense direction, are present before the first round of host gene transcription suggesting they may inform about the mechanisms shaping the first embryonic transcriptome. Dr. Eviskov is now taking experimental approaches to explore the effects of retrotransposons on the changing architecture of the embryonic genome.
Dr. Mimi de Vries developed a cre/loxP-based strategy to eliminate specific maternal mRNAs from the oocyte to determine their function during the oocyte-to-embryo transition. Using this technique she created a truncated form of beta-catenin, lacking the N-terminal portion of the protein that interacts with E-cadherin and with members of the Wnt-pathway. However, oocytes and embryos from these eggs are healthy and viable. Using antibodies specific to the C-terminal portion of beta-catenin she found a truncated protein, localized in the nucleus, suggesting that beta-catenin, which is known to bind to certain nuclear proteins is important in the oocyte-embryo transition. We plan to co-localize these nuclear proteins using the 4Pi microscope.
Stem cell-to-malignant precursor transition.
Karen Fancher, a Functional Genomics graduate student (U Maine), compares gene expression in mammary glands sampled from maturity to tumor appearance in a mouse model of mammary cancer, C57BL/6J-Tg(WapTAg)1Knw (Waptag 1). These transgenic mice contain a hybrid viral oncogene, Simian Virus 40 large Tumor-antigen (Tag) under the control of the whey acidic protein promoter, which targets expression SV40 Tag expression to mammary gland luminal epithelial cells. At about 7 months of age, multiparous Waptag 1 females develop ductal carcinoma in situ (DCIS), a clinical pre-tumor stage, which progresses to mammary carcinoma, resembling papillary human breast cancer, by 12 months of age, and finally advances into invasive glandular to solid mammary adenocarcinoma.
Together with Gary Churchill, TJL, she performed microarray analysis of statistically significant gene changes comparing various stages of Waptag 1 tumorigenesis with age-matched control C57BL/6J (B6) females. This analysis revealed stage-specific expression of retrotransposons and up-regulation of histones at very early on in the tumorigenic process. Dr. Anne Peaston designed a study to genetically tag the specific stem cell from which these tumors likely derive. These cells will be employed to determine the relationship between expression of the SV40Tag oncogene, activation of retrotransposons and chromatin changes in the earliest stages of tumorigenesis.
Recent publications:
Knowles BB, Evsikov AV, deVries WN, Peaston AE, Solter D. 2003. Gene expression in the oocyte to embryo transition and into preimplantation embryogenesis. In: Epigenesis versus preformation during mammalian development. Philosophical Transactions of the Royal Society Series B 358:1381-1388.
Solter, D, Hiragi T, Evsikov AV, Moyer J, DeVries WN, Peaston A E, Knowles BB. 2004. Epigenetic mechanisms in early mammallian development. In: Cold Spring Harbor Symposium 69:1-8.
Evsikov AV, de Vries WN, Peaston A, Fancher K, Chen F, Radford E, Latham K, Blake J, Bult C, Solter D, Knowles BB. 2004. Systems biology of the 2-cell embryo. Cytogenet Gen Res 105:240-250.
Chen W, Masterman K-A, Basta S, Dimopoulos N, Knowles BB, Bennink JR, Yewdell JW. 2004. Cross-Priming of CD8 + T Cells to Viral and Tumor Antigens is a Robust Phenomenon. Europ J Immunol 34:194-9.
de Vries WN, Evsikov AV, Fancher K, Haac BE, Solter D, Kemler R, Knowles BB. 2004. Development of mouse preimplantation embryos lacking maternal b-catenin and E-cadherin: insights into zygotic genome activation. Development 131:4435-45.
Peaston AE, Evsikov AV, Graber J, de Vries WN, Solter D, Knowles BB. 2004. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Develop Cell 7: 597-606.
Kemler, R, Hierholzer A, Kanzler B, Kuppig S, Hansen K, Taketo M, de Vries WN, Knowles BB, Solter D. 2004. Stabilization of b-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast. Development 131:4435-45.
Mikaelian I, Blades N, Churchill GC, Fancher K, Knowles BB, Eppig JT, Sundberg JP. 2004. Classification of spontaneous and transgenic mammary neoplasms, Breast Can Res 6:668-79.
Mehlmann,LM, Saeki Y, Tanaka S,. Brennan TJ, Evsikov AV, Pendola F, Knowles BB, EppigJJ, Jaffe LA. 2004. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 306:1947-50.
Kemler, R, Hierholzer A, Kanzler B, Kuppig S, Hansen K, Taketo M, de Vries WN, Knowles BB, Solter D. 2004. Stabilization of b-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast. Development 131:5817-24.
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