Transcription Factors in Trophectoderm Differentiation

描述(申请人提供)：滋养细胞早期产生人绒毛膜促性腺激素(HCG)对于挽救黄体和维持早期妊娠孕酮的产生是必不可少的。这一提议背后的主要假设是，OCT-4、Ets-2、DLX-3和GATA-2这三个转录因子在滋养外胚层的初始分化以及关键的滋养层特异基因的表达中具有指导作用。我们已经注意到，其表达上调为滋养外胚层第一形式的基因，包括hCG亚单位和干扰素-tau的基因，被转录因子Oct-4沉默，该转录因子具有维持内细胞团全能状态的作用，并被ETS-2、DLX-3和GATA-2反式激活。这项提议有四个目标。首先是阐明Oct-4与CG-β启动子的直接结合是如何导致沉默的。我们将确定Oct-4的哪些结构域参与其中，为什么Oct-4结合位置与共识结合序列如此显著地不同，并确定当Oct-4与启动子相关时，哪些蛋白质与Oct-4相互作用。目标2是了解Oct-4是如何沉默hCG-α的。在这里，我们认为目标是GATA-2或相关的GATA因子。一个目的是证明这一假设，但我们也将确定其他转录因子，结合“早期”的人滋养层细胞，这可能是额外的Oct-4靶点，以及OCT-4结合位点下游的阿尔法-ACT位点的作用，指导沉默。目标3是确定0ct-4是否逆转ETS-2反式激活hCG-α和hCG-β基因启动子的能力，以及如果观察到这种抑制是如何实现的。最终目的(目标4)是了解Oct-4、Ets-2、DLX-3和GATA-2的表达与滋养层细胞向干细胞样前体细胞分化的相互关系。将采用两个主要的模型系统：i)HT-H畸胎癌细胞，它在培养过程中自发地从小的、增殖的、漂浮的前体细胞转化为产生hCG的较大的附着的终点细胞；ii)通过在培养基中添加BMP-4，人类胚胎干细胞(HT1)可以定向向滋养层细胞。还将对绒毛膜癌细胞和足月胎盘的细胞滋养细胞进行验证性实验，这些细胞似乎对应于早期的前体滋养细胞。这两种类型的细胞都可以被诱导分化为产生大量hCG的形式。总之，这些实验将提供关于为什么这么多胚胎在滋养层细胞分化时丢失的洞察。这些研究有望揭示胚胎干细胞向滋养外胚层转变的机制，滋养外胚层是一种紧接着床前的发育事件，也是hCG开始表达的时间。

DESCRIPTION (provided by applicant): 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 is 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.