Mechanisms of gene expression and recombination

转录由被称为增强子的DNA元件调控，其中转录因子与特定的DNA基序结合。在基因被激活之前，增强剂是可获得的；了解受控的可及性和活性增强剂的产生一直是这个实验室的兴趣所在。 NIP-B样蛋白(NIPBL)与MAU2相关，是粘附素复合体与DNA结合、高阶染色质和环挤出所必需的。我们发现，NIPBL固有无序区(IDR)可能负责调节转录调控，但不是整个染色质组织。为了分析转录改变的基础，通过质谱学确定了NIPBL蛋白伙伴的分析。初步数据表明，虽然NIPBL与转录机制的关键成员如转录因子(CNOT1、CNOT2、RUNX3、NFAT2、NFKB1)、RNA聚合酶II亚基和中介复合体亚基相互作用，但IDR的缺失选择性地削弱了这种相互作用。 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断裂中，导致增强的环挤出和对断裂DNA的绝缘。我们确定，由于末端连接不当，磷酸化缺陷细胞的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在激活的调控DNA上的组合作用。本财年，我们通过分析500个小鼠和人类原代细胞，结合转录因子基序、足迹、结合(CHIP-SEQ)、转录本(RNA-SEQ)和可及性(ATAC-SEQ)的图谱，探索了哺乳动物中转录因子的协同作用。我们发现了两个转录因子组，它们与大多数表达的因子共定位，形成了层级聚集图中的条纹。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(USFs)，由30个SP、KLF、EGR和ZBTB家族成员组成，它们在所有被分析的组织中识别重叠的富含GC的序列。基因敲除和单分子跟踪显示，超临界流体使共处一地的合作伙伴获得了可及性，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一组基因表达的手段。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，而是以爆发的形式发生。每个基因产生的核糖核糖核酸的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，但似乎启动子上的DNA序列信息决定了幅度，而猝发频率取决于增强子上的序列信息。通过对单个基因的研究，人们已经了解到了很多关于转录爆发的知识。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。为了解决这一不足，本财年我们开发了一个计算机模型来分析单细胞转录本。这种方法第一次提供了一个关于细胞群体爆发的全面视图，而不需要用荧光分子标记单个基因。我们的未来目标是使用这一策略，利用缺乏已知在转录中起关键作用的蛋白质的细胞来确定它们是否调节爆裂。 研究基因表达在临床上的一个重要方面是更好地了解发生在恶性细胞中的基因调控网络，如B细胞淋巴瘤。这将使我们能够确定能够导致创新治疗干预的新靶点。弥漫性大B细胞淋巴瘤(DLBCL)是美国最常见的侵袭性淋巴瘤。基于DLBCL来源的细胞系，我们建立了47个关键B细胞基因的报告细胞系，并结合荧光激活细胞分选进行全基因组CRISPR/Cas9筛选以确定基因表达的调控因子。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，转录促进了抗体可变基因与D和J片段的重组，从而产生抗体的V(D)J部分。本财年，我们的实验室继续探索这些基因的转录如何促进V、D和J片段在重组过程中的物理配对。 在这个项目中，我们开发了一种全基因组的方法和软件套件来从单细胞RNA测序数据中推断转录动力学。我们将这种方法应用于多种细胞类型和扰动，发现：调控基因比管家基因更频繁地爆发，爆发的频率由介体(MED26)调节，爆发的大小由MYC控制，染色质结构蛋白(粘附素和CTCF)以及BRD4可以调节这两个参数。粘附素、BRD4和MED26在转录过程的不同阶段起作用；值得注意的是，MED26作用于染色质空间结构和预起始复合体的下游，与BRD4一起启动转录爆发。

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.