The development of an adult multi-cellular organism from a single fertilized egg requires the proliferation and differentiation of a large number of cells. In many species, the early post-fertilization divisions occur rapidly and synchronously without growth phases and cell cycle checkpoints. These early embryos are almost entirely transcriptionally inactive and therefore driven by maternally supplied RNAs. At the Mid-Blastula Transition (MBT), the embryo initiates large-scale transcription from the zygotic genome and cells gain growth phases and checkpoints. Previous work suggested that the MBT is initiated by the increased DNA-to-cytoplasmic ratio resulting from repeated rounds of DNA replication and cell division without cell growth. This led to the hypothesis that the progressive titration of an inhibitory factor present in the embryo allows the initiation of zygotic transcription. To understand the nature of this inhibitory activity we replicated early studies, which had been performed in intact embryos, in Xenopus laevis egg extracts and showed similar DNA concentration dependent transcriptional repression. Using this cell free system we have shown that adding DNA-coated beads to extracts lowers the threshold DNA concentration for transcriptional activation indicating that DNA beads titrate the inhibitory activity. Pre-incubatio of DNA-coated beads in egg extract reduces the beads'ability to induce transcription when the beads are added to a naive extract. Thus we are now able to biochemically assay and quantify the inhibitory activity. Using this assay we propose to biochemically purify the inhibitory factor() from Xenopus egg extract and to understand how the factor(s) contributes to transcriptional induction during development. )

Public Health Relevance

Identification of the control mechanism for zygotic transcription should provide a platform for the long-term understanding of gene expression in the embryo. As misregulated transcription may yield developmental defects, a molecular understanding of the MZT may give insight into epigenetic reprogramming and human disease.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HD073772-01A1
Application #
8508726
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Ravindranath, Neelakanta
Project Start
2013-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$235,143
Indirect Cost
$85,143
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305