Mechanisms of gene expression and recombination

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。在基因被打开之前，增强子变得可访问;了解活性增强子的可控可访问性和生成一直是本实验室的长期兴趣。 与MAU 2相关的Nipped-B样蛋白（NIPBL）是粘附素复合物与DNA和高阶染色质结合以及环挤出所必需的。使用基于CRISPR-Cas9的基因编辑，在CH 12 B细胞中产生NIPBL IDR缺失（NIPBL-IDRdel）突变体。我们发现，NIPBL固有无序区（IDR）可能是负责调谐转录调控，但不是整体染色质组织。为了剖析转录改变的基础，通过质谱法测定NIPBL蛋白伴侣的分析。 初步数据显示，虽然NIPBL与转录机制的关键成员相互作用，如转录因子（hocT 1，hocT 2，RUNX 3，NFAT 2，NFKB 1），RNA聚合酶II亚基和介体复合物亚基，IDR的缺失选择性地削弱这种相互作用。 今年我们还研究了粘附素对DNA修复的影响。我们发现，粘附素环通过磷酸化被招募到DNA断裂处，导致增强的环挤出和断裂DNA的绝缘性增加。我们确定磷酸化缺陷的细胞由于末端连接不正确而导致DNA修复延迟，导致易位和顺式缺失 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。本财政年度，我们通过分析500个小鼠和人类原代细胞，结合转录因子基序，足迹，结合（ChIP-seq），转录组（RNA-seq）和可访问性（ATAC-seq）的图谱，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。 研究基因表达的一个临床重要方面是更好地理解恶性细胞（如B细胞淋巴瘤）中发生的基因调控网络。这将使我们能够确定新的目标，可以导致创新的治疗干预措施。弥漫性大B细胞淋巴瘤（DLBCL）是美国最常见的侵袭性淋巴瘤类型。基于DLBCL衍生的细胞系，我们建立了47个关键B细胞基因的报告细胞系，并进行了全基因组CRISPR/Cas9筛选，结合荧光激活细胞分选，以鉴定基因表达的调节因子。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。 在这个项目中，我们开发了一个全基因组的方法和软件套件，从单细胞RNA测序数据推断转录动力学。我们将这种方法应用于多种细胞类型和扰动，并发现：调节基因比管家基因更频繁地爆发，爆发的频率由介质（MED 26）调节，爆发大小由MYC控制，染色质结构蛋白（cohesin和CTCF）以及BRD 4可以调节这两个参数。粘附素、BRD 4和MED 26在转录过程的不同阶段起作用;值得注意的是，MED 26在染色质空间结构和前起始复合物的下游起作用，与BRD 4一起工作，以启动转录爆发。

Transcription is regulated by DNA elements known as enhancers, where transcription factors bind to specific DNA motifs. Before genes are turned on, enhancers become accessible; understanding the controlled accessibility and generation of active enhancers has been a longstanding interest of this lab. Nipped-B-like protein (NIPBL), associated with MAU2, is required for the association of the cohesin complex with DNA and higher order chromatin and loop extrusion.Using CRISPR-Cas9-based gene editing, NIPBL IDR deletion (NIPBL-IDRdel) mutants were generated in CH12 B cells. We found that the NIPBL intrinsically disordered region (IDR) is likely responsible for tuning transcriptional regulation but not overall chromatin organization. To dissect the basis of the transcriptional alterations, analysis of NIPBL protein partners was determined via mass spectroscopy. Preliminary data reveal that while NIPBL interacts with key members of transcription machinery such as transcription factors (CNOT1, CNOT2, RUNX3, NFAT2, NFKB1), RNA Polymerase II subunits and Mediator complex subunits, deletion of the IDR selectively impairs this interaction. This year we also examined the impact of cohesin on DNA repair. We found that cohesin rings are recruited to DNA breaks through phosphorylation, leading to enhanced loop extrusion and increased insulation of the broken DNA. We determined that phosphorylation deficient cells were delayed in DNA repair with improper end joining, resulting in translocations and cis deletions 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 transcription factor 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. 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. A clinically important aspect of studying gene expression is to better understand gene regulatory networks occurring in malignant cells, such as B cell lymphoma. This will allow us to identify novel targets that can lead to innovative therapeutic interventions. Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive lymphoma in the US. Based on a DLBCL derived cell line, we established reporter cell lines for 47 key B cell genes and performed genome-wide CRISPR/Cas9 screening coupled to fluorescence-activated cell sorting to identify regulators of gene expression. 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. In this project, we have developed a genome-wide method and software suite to infer transcriptional kinetics from single-cell RNA sequencing data. We applied this approach across multiple cell types and perturbations and found that: Regulatory genes burst more frequently than housekeeping genes, the frequency of bursting is regulated by the mediator (MED26), burst size is governed by MYC, and chromatin architecture proteins (cohesin and CTCF) as well as BRD4 can modulate both parameters. Cohesin, BRD4 and MED26 act at different stages of the transcription process; notably, MED26 functions downstream of both chromatin spatial architecture and the preinitiation complex, working with BRD4, to initiate transcriptional burst.