The Role of the De-condensed Structure of Nascent Chromatin During T Cell Differentiation

项目摘要： 在细胞分化过程中，转录程序发生了变化，然后必须在细胞分化过程中维持转录程序。 依次基于染色质的表观遗传机制是维持和转换的核心， 转录程序。表观遗传标记的性质和遗传标记的基本问题， 在分化过程中转换这种标记的机制仍然不清楚，因为缺乏相关的 实验方法。我们开发了新的实验范式，可以研究 在单细胞和基因特异性水平上DNA复制过程中染色质的结构。使用我们的新 技术，我们发现显着差异的染色质结构在分化的过程中， 多能人类胚胎干细胞（hESC）和抗原无经验（幼稚）T细胞。期间 在诱导hESC分化为多巴胺神经元谱系或T细胞分化为多巴胺神经元谱系后的前几个小时， 在不同的T细胞亚群中，H3 K27 me 3在新生DNA上的积累显著延迟。以来 H3 K27 me 3在基因组中的出现与核小体的致密结构相一致， 提示在新生DNA上存在暂时解凝聚的核小体结构 诱导细胞分化后。我们的初步数据表明， 染色质的变性可能是谱系特异性转录因子（TF）向DNA募集所必需的 在细胞分化过程中诱导转录程序变化所必需的。所以我们 这些结果从分子上解释了被压抑的基因组的广大区域是如何被激活的 在细胞分化过程中。本提案的目标是使用不同的方法来测试两个独特的假设。 分化成多能hESC和特化T细胞的各种谱系的模型：1） 检查早期分化细胞中复制后染色质“开放”的时期是否是以下原因的结果： 几种组蛋白修饰蛋白活性的复杂相互作用;和2）为了检查这是否 开放的复制后染色质为谱系的高度可及性创造了“机会之窗”- 指定在细胞分化期间改变转录程序所需的TF。 检查这些独特的假设可能提供一个普遍的基于染色质的分子机制， 细胞的生物可塑性。

Project Summary: During cell differentiation, transcriptional programs are changed, and then must be maintained in turn. Chromatin-based epigenetic mechanisms are at the core of maintenance and switching of transcriptional programs. The fundamental issues of the nature of epigenetic marking and of the mechanisms that switch this marking during differentiation remain unclear due to lack of relevant experimental approaches. We developed new experimental paradigms that allow investigating the structure of chromatin during DNA replication at a single-cell and at a gene-specific levels. Using our new techniques, we found striking differences in the structure of chromatin during differentiation of the pluripotent human embryonic stem cells (hESC) and the antigen-inexperienced (naïve) T cells. During the first several hours after induction of differentiation of hESCs to dopamine neuron lineage, or T cells to different T cell subsets, accumulation of H3K27me3 is significantly delayed on nascent DNA. Since the occurrence of H3K27me3 in the genome coincides with the dense structure of nucleosomes, this suggests the existence of a temporarily de-condensed structure of nucleosomes on nascent DNA shortly after induction of cell differentiation. Our preliminary data indicate that the de-condensed, `open' structure of chromatin may be essential for recruitment to DNA of the lineage-specific transcription factors (TFs) that are essential to induce changes in transcriptional programs during cell differentiation. Thus, our results present a molecular explanation of how the vast areas of the repressed genome can be activated during cell differentiation. The goals of this proposal are to test two unique hypotheses using different models of differentiation to various lineages for pluripotent hESCs and for specialized T cells: 1) To examine whether the period of `open' post-replicative chromatin in early differentiating cells is a result of complex interplay of activities of several histone-modifying proteins; and 2) To examine whether this open post-replicative chromatin creates a `window of opportunity' for high accessibility of lineage- specifying TFs that are required to change the transcriptional program during cell differentiation. Examining these unique hypotheses may provide a universal chromatin-based molecular mechanism for biological plasticity of the cell.