Role of Germ Cell Nuclear Factor in Restricting hESC Pluripotency

多能基因，如Oct4和Nanog，对于维持自我更新和 在ES细胞中分化为所有类型的细胞。在ES细胞分化抑制和沉默过程中 多能基因伴随着早期谱系决定因素的表达而出现。我们有 研究表明，孤儿核受体GCNF(生殖细胞核因子)在 通过结合Oct4和Nanog启动子靶点抑制多潜能基因的表达。GCNF“‘”MES细胞 未能抑制多潜能基因的表达，因此在没有白血病的情况下茁壮成长 抑制因子(LIF)。初步数据表明，除了沉默多能基因外，GCNF 在早期分化过程中限制谱系决定因素的早熟表达。它的作用机制 抑制涉及GCNF与多个共抑制复合体的相互作用，包括核受体 共抑制子(NCoR)，以及DNA甲基化机制的多个组件，包括DNA 甲基转移酶和甲基结合结构域(MBD)因子，它们是组蛋白的组成部分 脱乙酰基复合体。由于它在抑制ES细胞基因表达方面起着关键作用，因此至关重要的是 我们更好地了解了这种抑制因子在调节HES细胞多能性中的分子作用。这个 我们要检验的假设是，GCNF在分化过程中建立了一个瞬时窗口 人类ES细胞，通过抑制谱系决定因素的早期表达，从而允许有序 通过招募多种共抑制物靶向抑制多潜能基因表达 基因启动子。为了验证这一假设，我们有以下目的：(1)定义hGCNF在 抑制HES细胞分化过程中已知的靶基因，如Oct4、Nanog和Cripto。在……里面 此外，GCNF和P53在抑制Nanog表达方面的功能协同作用将是 与项目3合作研究。(2)确定GCNF在抑制多能性基因中的作用 和HES细胞分化过程中的谱系标志物。一种全基因组的方法来定义对 人胚胎干细胞分化过程中的基因表达。我们将使用非对称微阵列的组合 分析和ChlP芯片实验，以确定GCNF直接和间接调控的基因。 这些概况将与项目3和4中获得的P53和THAP11的概况进行比较。(3)定义 HES细胞分化过程中基因沉默的GCNF依赖机制。GCNF相互作用因素 将使用生化方法和候选辅阻遏子的招募从HES细胞中分离出 TO多能性启动子将在HES细胞分化过程中进行分析，并与时间相关 在这些启动子上发生的表观遗传变化。我们还将寻找与THAP11的特定交互作用， P53和NANOG(项目1、3和4)。。

Pluripotent genes, such as Oct4 and nanog are essential to maintain self-renewal and the ability to differentiate into all cell types in ES cells. During ES cell differentiation repression and silencing of pluripotent genes occurs concomitantly with' the the expression of early lineage determinants. We have shown that the orphan nuclear receptor GCNF (Germ Cell Nuclear Factor) plays a pivotal role in the repression of pluripotent gene expression by binding Oct4 and nanog promotor targets. GCNF"'" mES cells fail to repress pluripotent gene expression and as a consequence grow robustly in the absence of leukemia inhibitory factor (LIF). Preliminary data suggest that in addition to silencing pluripotency genes, GCNF restricts precocious expression of lineage determinants during early differentiation. The mechanism of repression involves GCNF interactions with multiple co-repressor complexes, including the nuclear receptor co-repressor (NCoR), and with multiple components of the DNA methylation machinery, including DNA methyltransferases and Methyl Binding Domain (MBD) factors, which are components of histone deacetylation complexes. Because of its pivotal role in repression of ES cell gene expression, it is critical that we better understand the molecular role of this represser in the regulation of hES cell pluripotency. The hypothesis that we will test is that GCNF establishes a transient window during differentiation of human EScells, by inhibiting early expression of lineage determinants, which permits orderly repression of pluripotent gene expression by recruiting various co-repressor complexes to target gene promoters. To test this hypothesis, we have the following aims: (1) define the role of hGCNF in repression of known target genes during hES cell differentiation, such as Oct4, nanog and Cripto. In addition, functional synergy between GCNF and p53 in the repression of Nanog expression will be investigated in collaboration with Project 3. (2) define the role of GCNF in repression of pluripotency genes and lineage markers during hES cell differentiation. A genome-wide approach to defining the regulation of gene expression during human ES cell differentiation. We will use a combination of Affymetric microarray analyses and ChlP-on-chip experiments to define the genes directly and indirectly regulated by GCNF. These profiles will be compared with those obtained for p53, and THAP11 in Projects 3 and 4. (3) define the GCNF-dependent mechanism of gene silencing during hES cell differentiation. GCNF interacting factors will be isolated from hES cells using a biochemical approach and the recruitment of candidate corepressors to pluripotency promoters will be analyzed during hES cell differentiation and correlated with temporal epigenetic changes occurring at these promoters. We will also look for specific interactions with THAP11, p53, and NANOG (with Projects 1, 3, and 4). .