Mechanisms of gene expression and recombination

转录调控是细胞协调单个基因或基因组表达的手段。最近对单细胞的研究表明，在大多数情况下，转录不是一种连续的活动，而是以爆发的方式发生的。每个基因产生的RNA量与这种爆发的幅度和频率成正比。这两个参数是如何控制的还不完全清楚，但似乎启动子的DNA序列信息决定了振幅，而爆发频率依赖于增强子的序列信息。通过对单个基因的研究，我们已经对转录爆发有了很多了解。然而，这样的实验是耗时的，并且不能提供细胞群体或组织中基因表达的全局视图。为了解决这一缺陷，本财年我们开发了一个计算机模型来分析单细胞转录组。这种方法第一次提供了一个全面的观点，在细胞群中的爆裂，而不需要标记单个基因与荧光分子。我们未来的目标是使用这种策略与已知在转录中起关键作用的蛋白质缺乏的细胞，以确定它们是否调节爆发。 转录由称为增强子的DNA元件调节，其中转录因子与特定的DNA基序结合。在基因启动之前，通过去除DNA上的甲基，增强子变得可访问。这种活性已被证明会引起DNA损伤。本财政年度，我们与Andre Nussenzweig（NCI）合作表明，神经元的这种损伤可能是人类神经退行性疾病发展的原因。 转录调控的一个重要特征是转录因子对活性调控DNA的组合作用。本财年，我们通过分析500个小鼠和人类原代细胞，结合TF基序、足迹、结合（ChIP-seq）、转录组（RNA-seq）和可及性（ATAC-seq）图谱，探索了哺乳动物中转录因子的协同性。我们发现了两个转录因子群，它们与表达最多的因子共定位，在分层聚类图中形成条纹。第一组包括广泛占据DNA元件的谱系决定因子，这与它们在组织特异性转录中的关键作用一致。第二个，被称为通用条纹因子（USF），包括30个SP，KLF，EGR和ZBTB家族成员，在所有分析的组织中识别重叠的富含GC的序列。敲除和单分子跟踪显示，USF赋予了共定位的合作伙伴的可访问性，并增加了他们的停留时间。 转录的另一个重要特征是它控制成熟B细胞中的重组。例如，在骨髓中，转录促进抗体可变基因与D和J区段的重组以产生抗体的V（D）J部分。本财政年度，我们的实验室继续探索这些基因的转录如何在重组过程中促进V，D和J片段的物理配对。

Transcriptional regulation is the means whereby cells orchestrate expression of individual genes or group of genes. Recent studies with single cells have shown that, for the most part, transcription is not a continuous activity but it occurs in bursts. The amount of RNA produced for each gene is directly proportional to the amplitude and frequency of such bursts. How these two parameters are controlled is not totally clear, but it appears that DNA sequence information at promoters determine the amplitude, while burst frequencies rely on sequence information at enhancers. Much has been learned on transcriptional bursting through the study of individual genes. However such experiments are time consuming and do not provide a global view of gene expression in cell populations or tissues. To address this deficiency, this fiscal year we have developed a computer model to analyze single cell transcriptomes. This approach provides for the first time a comprehensive view of bursting in a cell population without the need to label individual genes with fluorescent molecules. Our goal moving forward is to use this strategy with cells deficient for proteins known to play key roles in transcription to determine whether they regulate bursting. Transcription is regulated by DNA elements known as enhancers, where transcription factors bind to specific DNA motifs. Before genes are turn on, enhancers become accessible through the removal of methyl groups on DNA. This activity has been shown to induce DNA damage. This fiscal year we have shown in collaboration with Andre Nussenzweig (NCI) that such damage in neurons may be responsible for the development of neurodegenerative diseases in humans. An important feature of transcription regulation is the combinatorial action of transcription factors at active regulatory DNA. This fiscal year, we explored transcription factor cooperativity in mammals by analyzing 500 mouse and human primary cells, combining an atlas of TF motifs, footprints, binding (ChIP-seq), transcriptomes (RNA-seq), and accessibility (ATAC-seq). We uncovered two transcription factor groups that colocalized with most expressed factors, forming stripes in hierarchical clustering maps. The first group included lineage-determining factors that occupied DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprised 30 SP, KLF, EGR, and ZBTB family members that recognized overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking revealed that USFs imparted accessibility to colocalized partners and increased their residence time. Another important feature of transcription is its control of recombination in maturing B cells. In the bone marrow for instance, transcription facilitates the recombination of antibody variable genes to D and J segments to produce the V(D)J portion of antibodies. This fiscal year, our laboratory has continue exploring how transcription of these genes facilitate the physical pairing of V, D and J segments during recombination.