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FUNCTIONAL GENOMICS AND MECHANISM OF BCL11B ACTION IN LYMPHOCYTE COMMITMENT

BCL11B 在淋巴细胞定型中的作用基因组学和机制

基本信息

批准号：
10393519
负责人：
ELLEN V. ROTHENBERG
金额：
$ 63.23万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-04 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10393519
关键词：
3-Dimensional Acute Affect Binding Binding Sites Biological CRISPR/Cas technology Cell Lineage Cells ChIP-seq Chromatin Chromatin Loop Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA DNA Binding Defect Development Developmental Gene Developmental Process Diagnosis Disease Dissection E protein Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Predisposition to Disease Genetic Transcription Genetic study Genome Genome Mappings Genomic Segment Genomic approach Genomics Human Genetics ID2 gene Immune Immune system Investments Knock-out Knockout Mice Light Link Location Lymphocyte Lymphoid Cell Maps Mature T-Lymphocyte Mediating Molecular Natural Killer Cells Nuclear Hormone Receptors Output Pattern Property Proteomics Radiation Regulation Regulator Genes Research Personnel Resolution Resources Role Site System T-Cell Development T-Cell Lymphoma T-Lymphocyte T-Lymphocyte Subsets Techniques Testing Time Transcriptional Regulation Work Zinc Fingers alpha-beta T-Cell Receptor cell type chicken ovalbumin upstream promoter-transcription factor cofactor functional genomics gene network genetic corepressor genome-wide leukemia loss of function preference protein complex protein function recruit retroviral transduction transcription factor

项目摘要

ABSTRACT/PROJECT SUMMARY Bcl11b is a bifunctional zinc finger transcription factor that is crucial for the lineage commitment of TCRαβ T cells. The Bcl11b gene is activated precisely at the developmental transition when the cells undergo commitment, and it sustains αβ T-cell identity and functions of multiple T-cell subsets thereafter. Genes that it keeps silent during initial T-cell commitment include key regulators of all innate lymphoid cells (ILCs) and natural killer cells (NK). Some of these genes are also continuously repressed by Bcl11b in mature T cells. However, surprisingly, Bcl11b is also expressed in one particular class of innate lymphoid cells, ILC2 cells. ILC2 cells also require Bcl11b for their identity, yet at the same time, they express Bcl11b along with Id2 and other genes that Bcl11b normally silences in T cells. A major question is how Bcl11b works so differently in ILC2 and early developing T cells (pro-T cells). We propose to determine how this works using functional genomics and gene network dissection to shed light on the mechanisms that make the difference. Our preliminary evidence from ChIP-seq and proteomic analyses shows that Bcl11b binds to distinct, only partially overlapping sets of sites in the two cellular contexts, and interacts with different transcriptional partners; and this is correlated with regulation of almost completely nonoverlapping sets of Bcl11b-sensitive genes in the two contexts. To interpret these differences, we first distinguish functional from probably-nonfunctional sites where Bcl11b binds DNA, by identifying its impact on complexes of co-regulators that assemble with it. Thus, the subset of sites is mapped where Bcl11b has a locally indispensable nucleation role for chromatin-modulating cofactors and/or other specific interaction partners. Second, we plan to map genomic regions where higher-order chromatin looping interactions and compartment boundaries can also be shown to depend on Bcl11b expression. These results will identify the Bcl11b binding sites in each cell context where that binding is molecularly functional. To determine the underlying causes of these differences, we will test how Bcl11b's own site binding preferences may be established through the presence of other transcription factors. We have identified at least two other important sequence-specific transcription factors, e.g., that (1) interact extensively with Bcl11b at many of its binding sites in the T-cell context, yet (2) are under-represented in ILC2 cells. Using retroviral transduction and CRISPR technology adapted to pro-T cells, we will introduce or delete putative interaction partners reciprocally in ILC2 and pro-T cells, to evaluate critically what roles they may have in guiding the binding and function of Bcl11b. We will apply this approach to explain how Bcl11b nucleates functionally different sites in pro-T cells and ILC2s. Finally, an interaction of particular importance is the gene network circuit through which we have shown Bcl11b to support E proteins in establishing the T-lineage commitment regulatory state. Using conditional double knockouts, we will resolve the subsets of Bcl11b gene regulation effects that are mediated through this circuit from those that are controlled directly.

摘要/项目摘要 Bcl11b是一种双功能锌指转录因子，对TcRαβT的谱系承诺至关重要细胞。Bcl11b基因正是在细胞经历发育转变时被激活的承诺，并维持αβ的T细胞身份和多个T细胞亚群的功能。基因表明它在最初的T细胞承诺期间保持沉默包括所有天然淋巴样细胞(ILCs)和自然细胞的关键调节因子杀伤细胞(NK)。在成熟的T细胞中，其中一些基因也被Bcl11b持续抑制。然而，令人惊讶的是，Bcl11b也在一类特殊的固有淋巴样细胞ILC2细胞中表达。ILC2细胞也需要Bcl11b作为其身份，但同时，它们表达Bcl11b和Id2等基因 Bcl11b通常在T细胞中沉默。一个主要问题是Bcl11b在ILC2和早期版本中的工作方式有何不同发育中的T细胞(原T细胞)。我们建议使用功能基因组学和基因技术来确定这是如何起作用的网络剖析，以阐明造成差异的机制。我们的芯片序列和蛋白质组分析的初步证据表明，bcl11b只与不同的在两种细胞环境中部分重叠的一组位点，并与不同的转录伙伴相互作用；这与对Bcl11b敏感基因集几乎完全不重叠的调节有关有两个背景。为了解释这些差异，我们首先区分功能站点和可能的非功能站点通过确定Bcl11b对与其组装的协同调节分子复合体的影响，Bcl11b与DNA结合的位置。因此，在Bcl11b对染色质调节具有局部不可缺少的成核作用的位置定位协同因素和/或其他特定交互伙伴。第二，我们计划绘制基因组区域的更高顺序染色质环相互作用和间隔边界也可以被证明依赖于Bcl11b的表达。这些结果将确定每个细胞环境中的Bcl11b结合位点，其中该结合是分子上的功能齐全。为了确定这些差异的根本原因，我们将测试Bcl11b自己的站点绑定偏好可以通过其他转录因子的存在来建立。我们已经确认了至少另外两个重要的序列特异性转录因子，例如：(1)与Bcl11b在许多地方广泛相互作用在T细胞环境中，(2)在ILC2细胞中表达不足。利用逆转录病毒转导和适用于PRO-T细胞的CRISPR技术，我们将引入或删除假定的相互作用伙伴在ILC2和PRO-T细胞中相互作用，以批判性地评估它们在指导结合和 Bcl11b的功能。我们将应用这种方法来解释Bcl11b是如何在 PRO-T细胞和ILC2。最后，一个特别重要的相互作用是基因网络电路，通过它我们已经证明Bcl11b在建立T谱系承诺调控状态时支持E蛋白。vbl.使用条件性双敲除，我们将解决Bcl11b基因调控效应的亚集通过这个电路从那些被直接控制的。