Repression via Facultative Heterochomatin

 描述(申请人提供)：哺乳动物细胞分化的复杂程序最终以染色质为目标，形成可进入或偏离转录机制的染色质环境，从而有助于不同的基因表达谱。如何首先建立这些染色质结构来设定转录程序，以及如何在细胞分裂期间将这些已建立的结构恢复到新复制的DNA上，这是表观遗传学的关键。基于上一次资助期间的初步发现，我们将全面探索我们已广泛研究的表观遗传信息的两个主要调控因子：PrC1和PrC2，以及第三个潜在的表观遗传调控因子CTCF。我们将继续研究它们针对基因组离散区域的特异性的分子基础，控制它们活性的动态，以及它们的活动如何传递适当的转录输出。由Polycomb Group蛋白组成的两个哺乳动物复合体PRC1和PRC2是公认的转录抑制的表观遗传转运体。Prc1通过催化组蛋白H_2A在赖氨酸119上的单泛素化和染色质紧致来传递阻遏作用。通过我们广泛的生化分析，我们证明了PRC1包含了离散的但不同的复合体。在目标1中，我们扩展了我们的初步结果，表明一些PRC1亚组的区别蛋白要么像神经元特异性蛋白AUTS2那样将PRC1转化为转录激活因子，要么像心肌细胞中的FBRSL1那样表现出细胞类型特异性和可能的新的抑制机制，或者像运动神经元丰富的YAF1那样调节PRC1向染色质的募集。在AUTS2和FBRSL1的情况下，将分别使用生化和基因组方法以及小鼠模型来研究特定基因表达的潜在机制基础和结果。在目标2中，我们继续我们对PRC2的广泛分析，它催化组蛋白H3在赖氨酸27处的甲基化，这是一种抑制染色质的修饰。我们利用生化分析和CRISPR技术探索了调控PRC2活性的参数，包括其催化亚基Ezh2的翻译后修饰，以及自然发生的显性负性组蛋白突变体的负面影响。在我们对长非编码RNA与Ezh2和PRC2相关蛋白Jarid2相互作用的研究基础上，我们扩展到这些相互作用在介导PRC2向染色质招募的特异性中的作用。在目标3中，我们利用CHIP-SEQ、序列捕获Hi-C和CHIA-PET技术，探索了RNA介导的CTCF多聚化及其在CTCF介导的HOX基因簇内染色质边界调节中的可能作用。

 DESCRIPTION (provided by applicant): The intricate programs of mammalian cell differentiation ultimately target chromatin, formulating chromatin environments accessible to or deflective of the transcriptional machinery and thus conducive to distinct gene expression profiles. How these chromatin structures are first established to set the transcription program and how these established structures are then re-instated on newly replicated DNA during cell division is the crux of epigenetics. Based on preliminary findings obtained during the previous funding period, we will comprehensively explore two major modulators of epigenetic information that we have investigated extensively: PRC1 and PRC2, and a third potential epigenetic modulator, CTCF. We will continue our investigation of the molecular basis of their specificity in targeting discrete regions of the genome, the dynamics controlling their activity, and how their activities convey appropriate transcription outputs. Two mammalian complexes that comprise Polycomb Group proteins, PRC1 and PRC2 are recognized epigenetic conveyers of transcriptional repression. PRC1 transmits repression through catalysis of monoubiquitination of histone H2A at lysine 119 and chromatin compaction. Through our extensive biochemical analyses, we demonstrated that PRC1 embodies discrete yet heterogenous complexes. In aim 1, we expand on our preliminary results showing that the distinguishing proteins for some of the PRC1 subgroups either convert PRC1 into a transcriptional activator, as in the case of the neuronal-specific protein, AUTS2, or exhibit cell-type specificity and a possible novel repressive mechanism as in the case of FBRSL1 in cardiomyocytes, or modulate PRC1 recruitment to chromatin as in the case of motor neuron enriched YAF1. The underlying mechanistic basis and the outcome to specific gene expression will be studied using biochemical and genomic approaches, respectively and with mouse models in the case of AUTS2 and FBRSL1. In aim 2, we continue our extensive analyses of PRC2 that catalyses methylation of histone H3 at lysine 27, a modification of repressive chromatin. We explore parameters regulating PRC2 activity including post-translational modifications of its catalytic subunit Ezh2, and the negative effects of naturally occurring, dominant negative histone mutants using biochemical analyses and CRISPR technology. With the foundation of our studies of interactions of long noncoding RNA with both Ezh2 and the PRC2 associated protein Jarid2, we expand into the role of these interactions in mediating specificity in PRC2 recruitment to chromatin. In aim 3, we pursue our preliminary results of RNA-mediated CTCF multimerization and its possible role in CTCF- mediated regulation of chromatin boundaries within the HOX gene cluster using ChIP-seq, sequence capture Hi-C and ChIA-PET technologies.