Role of Germ Cell Nuclear Factor in Restricting hESC Pluripotency

多能基因，如Oct4和nanog，对维持自我更新和自我更新的能力至关重要。 在胚胎干细胞中分化成所有细胞类型。在ES细胞分化过程中， 多能基因与早期谱系决定因子的表达同时发生。我们有 显示孤儿核受体GCNF（生殖细胞核因子）在生殖细胞的发育中起着关键作用。 通过结合Oct4和nanog启动子靶抑制多能基因表达。GCNF-mES细胞 不能抑制多能基因表达，因此在没有白血病的情况下生长健壮 抑制因子（LIF）。初步数据表明，除了沉默多能性基因，GCNF 限制了早期分化过程中谱系决定因素的早熟表达。的机理 阻遏涉及GCNF与多种辅阻遏物复合物的相互作用，包括核受体 辅阻遏物（NCoR），并与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，它们是组蛋白的组分 脱乙酰化络合物。由于其在抑制ES细胞基因表达中的关键作用， 我们更好地理解了这种阻遏物在调节hES细胞多能性中的分子作用。的 我们将检验的假设是，GCNF在分化过程中建立了一个短暂的窗口， 人ES细胞，通过抑制谱系决定簇的早期表达， 通过募集各种共阻遏物复合物靶向多能基因表达的阻遏 基因启动子为了验证这一假设，我们有以下目标：（1）确定hGCNF在以下方面的作用： 在hES细胞分化过程中抑制已知的靶基因，如Oct4、nanog和Cripto。在 此外，GCNF和p53在抑制Nanog表达中的功能协同作用将被证实。 与项目3合作。(2)确定GCNF在抑制多能性基因中的作用 和谱系标志物。一种全基因组的方法来定义 人ES细胞分化过程中的基因表达。我们将使用Affymetric微阵列 分析和ChIP芯片实验以确定由GCNF直接和间接调节的基因。 这些曲线将与项目3和4中p53和THAP 11获得的曲线进行比较。(3)定义 hES细胞分化过程中GCNF依赖的基因沉默机制。GCNF相互作用因子 将使用生物化学方法和候选辅阻遏物的募集从hES细胞中分离 将在hES细胞分化期间分析多能性启动子的表达，并将其与时间相关性进行分析。 在这些启动子上发生的表观遗传变化。我们还将寻找与THAP 11的特定相互作用， 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). .