Chromatin Domain Structure and Function

转录调控是最基本的分子和细胞生物学过程的基础。我们的 长期目标是确定10 nm和30 nm染色质纤维如何折叠成大规模染色质结构域， 这些染色质结构域如何相对于特定的核体在细胞核内移动和定位 以及这对转录和复制等DNA功能意味着什么。 我们的第一个目标是剖析转录非依赖和转录依赖的机制 基因组相对于核斑点的位置。我们的理论基础是相对于核的基因定位 SPECKLES可能为数千个基因调节基因表达水平，因此剖析 这些机制将揭示基因调控的新方面。这一目标建立在强有力的前期工作基础上。 在上一个资助周期中，我们发现了核斑点接触后基因表达的放大。我们 还发现了一种令人惊讶的保守的基因组到核斑点的距离，只有很小的距离移动 相对斑点与基因表达的大变化和许多诱导基因高度相关 甚至在转录激活之前就“预先定位”在核斑点附近。 我们的第二个目标是绘制、可视化、重建，然后剖析大型- 染色质结构域具有不同程度的紧凑性；这些结构域可以通过活细胞观察到 显微镜和电子显微镜，但还没有用目前的基因组方法测量，如Hi-C和 通过传统的鱼类方法并不能很好地保存。我们的理由是，这一水平的大规模 压实调节转录起始和延伸率，因此我们对 染色质结构域具有不同程度的大规模染色质压缩将揭示基因的新方面 监管。这一目标还建立在强有力的前期工作的基础上。在上一个拨款周期内，我们采用了高斜坡。 TSA-SEQ分数来识别异常解浓缩的大规模染色质结构域(DLCD)。DLCD 主要映射到Hi-C舱、小舱和TAD边界，分隔活动和 抑制的染色质结构域。酸性激活剂和染色质因子被酸性激活剂招募 在数百种染色质修饰因子中，DLCDS的浓缩程度最高。令人惊讶的是，这观察到 浓缩与这笔赠款最初几年的结果相联系，表明在四类 转录激活剂，只有酸性转录激活剂结构域显示出共同的诱导活性 工程异染色质染色体区域的大规模解凝聚。在这最后一次拨款中 周期中，我们还在开发TSA-MS以识别定位于 免疫染色的核体以及大DNA结构的操纵和转基因。

Transcriptional regulation is fundamental to most basic molecular and cell biological processes. Our long-term goals are to determine how 10 and 30 nm chromatin fibers fold into large-scale chromatin domains, how these chromatin domains are moved and positioned within nuclei relative to specific nuclear bodies and compartments, and what this means for DNA functions such as transcription and replication. Our first Aim is to dissect both transcription independent and transcription dependent mechanisms for genome positioning relative to nuclear speckles. Our rationale is that gene positioning relative to nuclear speckles modulates levels of gene expression possibly for thousands of genes, and therefore dissection of these mechanisms will reveal novel aspects of gene regulation. This Aim builds on strong preliminary work. During the last grant cycle, we discovered gene expression amplification after nuclear speckle contact. We also discovered a surprising conservation of genome distance to nuclear speckles, with small distance shifts relative to speckles highly correlated with large changes in gene expression and with many inducible genes “pre-positioned” near nuclear speckles even before transcriptional activation. Our second Aim is to map, visualize, reconstitute, and then dissect cis and trans determinants for large- scale chromatin domains with distinct levels of compaction; these domains can be visualized by live-cell microscopy and electron microscopy but are not yet measured by current genomic methods such as Hi-C and are not well-preserved by conventional FISH methods. Our rationale is that this level of large-scale compaction modulates both transcriptional initiation and elongation rates and therefore our analysis of chromatin domains with different levels of large-scale chromatin compaction will reveal novel aspects of gene regulation. This Aim also builds on strong preliminary work. During the last grant cycle, we used high slopes of TSA-seq scores to identify unusually decondensed large-scale chromatin domains (DLCDs). DLCDs mapped predominately to Hi-C compartment, subcompartment, and TAD boundaries separating active and repressed chromatin domains. Acidic activators and chromatin factors recruited by acidic activators showed the highest enrichment over DLCDS among hundreds of chromatin-modifying factors. Strikingly, this observed enrichment connects with results from the early years of this grant showing that among four classes of transcriptional activators, only acidic transcriptional activator domains showed the common activity of inducing large-scale decondensation of an engineered heterochromatic chromosome region. During this last grant cycle, we also made technological advances in developing TSA-MS to identify proteins localizing to immunostained nuclear bodies and in the manipulation and transgenesis of large DNA constructs.