The early production of human chorionic gonadotropin (hCG) by the trophoblast is essential to rescuing the corpus luteum and maintaining production of progesterone in early pregnancy. The major hypothesis behind the proposal is that three transcription factors, Oct-4, Ets-2, Dlx-3 and GATA-2, have a directive role in the initial differentiation of trophectoderm, as well as in the expression of key trophoblast-specific genes. We have noted that genes whose expressions are upregulated as trophectoderm first forms, including those for the hCG subunits and interferon-tau, are silenced by the transcription factor Oct-4, which has a role maintaining the inner cell mass in a totipotent state, and transactivated by Ets-2, Dlx-3 and GATA-2. There are four aims to this proposal. The first is to elucidate how the direct binding of Oct-4 to the CG-beta promoter results in silencing. We shall determine what domains of Oct-4 are involved, why the Oct-4 binding site differs so markedly from a consensus binding sequence, and identify which proteins interact with Oct-4 when it is associated with the promoter. Goal 2 is to understand how Oct-4 silences hCG-alpha. Here, we believe that the target is GATA-2 or a related GATA factor.
One aim i s to prove this hypothesis, but we shall also identify other transcription factors that bind Oct-4 in """"""""early"""""""" human trophoblast cells, which could be additional Oct-4 targets, and the role of an Oct-4 binding site downstream of the alpha-ACT site where silencing is directed. Goal 3 is to determine whether 0ct-4 reverses the ability of Ets-2 to transactivate the hCG-alpha & hCG-beta gene promoters and how such an inhibition, if observed, is achieved.
The final aim (Aim 4) is to understand the interrelationships between expression of Oct-4, Ets-2, Dlx-3 and GATA-2 and the differentiation of trophoblast from stem cell-like precursors. Two main model systems will be employed: i) HT-H teratocarcinoma cells, which convert spontaneously during culture from small, proliferating, floating precursors to larger, attached end-point cells that produce hCG; ii), human embryonic stem cells (HT 1) that can be directed towards trophoblast by addition of BMP-4 to the culture medium. Confirmatory experiments will also be carried out with choriocarcinoma cells, which appear to correspond to early precursor trophoblast, and cytotrophoblast from term placenta. Both these types of cell can be induced to differentiate to forms that produce abundant hCG. Together, these experiments will provide insight into why so many embryos are lost at about the time that the trophoblast is differentiating. The studies are expected to reveal mechanisms underpinning the transition of embryonic stem cells to trophectoderm, a developmental event that immediately precedes implantation and a time when expression of hCG begins.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
3R01HD042201-04S1
Application #
7227276
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Tasca, Richard J
Project Start
2003-07-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
4
Fiscal Year
2006
Total Cost
$110,250
Indirect Cost
Name
University of Missouri-Columbia
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
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Ezashi, Toshihiko; Das, Padmalaya; Gupta, Rangan et al. (2008) The role of homeobox protein distal-less 3 and its interaction with ETS2 in regulating bovine interferon-tau gene expression-synergistic transcriptional activation with ETS2. Biol Reprod 79:115-24

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