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

• 批准号: 9914203
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-04 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9914203
• 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; genetic corepressor; genome-wide; leukemia; loss of function; preference; protein complex; protein function; recruit; retroviral transduction; transcription factor

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，对于 TCRαβ T 的谱系定型至关重要 细胞。 Bcl11b 基因在细胞经历发育转变时被精确激活。 承诺，并且此后它维持 αβ 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 细胞定向过程中保持沉默的因素包括所有先天淋巴细胞 (ILC) 和天然淋巴细胞的关键调节因子 杀伤细胞（NK）。其中一些基因也在成熟 T 细胞中被 Bcl11b 持续抑制。然而， 令人惊讶的是，Bcl11b 也在一类特定的先天淋巴细胞 ILC2 细胞中表达。 ILC2细胞也 需要 Bcl11b 来确定其身份，但同时，它们也表达 Bcl11b 以及 Id2 和其他基因，这些基因 Bcl11b 通常在 T 细胞中沉默。一个主要问题是 Bcl11b 在 ILC2 和早期的工作方式有何不同？ 发育中的 T 细胞（前 T 细胞）。我们建议使用功能基因组学和基因来确定其工作原理 网络剖析以揭示产生差异的机制。 我们来自 ChIP-seq 和蛋白质组学分析的初步证据表明，Bcl11b 仅与不同的结合 两个细胞环境中部分重叠的位点组，并与不同的转录伙伴相互作用； 这与 Bcl11b 敏感基因组几乎完全不重叠的调节相关 两个上下文。为了解释这些差异，我们首先区分功能性站点和可能非功能性站点 Bcl11b 与 DNA 结合，通过识别其对与其组装的共调节因子复合物的影响。因此， 绘制了 Bcl11b 位点子集，其中 Bcl11b 对染色质调节具有局部不可缺少的成核作用 辅助因子和/或其他特定的相互作用伙伴。其次，我们计划绘制高阶基因组区域图谱 染色质环相互作用和区室边界也取决于 Bcl11b 表达。 这些结果将识别每个细胞环境中的 Bcl11b 结合位点，其中该结合是分子上的 功能性的。为了确定这些差异的根本原因，我们将测试 Bcl11b 自身的位点结合方式 偏好可以通过其他转录因子的存在来建立。我们至少确定了 另外两个重要的序列特异性转录因子，例如 (1) 在许多方面与 Bcl11b 广泛相互作用 其在 T 细胞环境中的结合位点，但 (2) 在 ILC2 细胞中代表性不足。使用逆转录病毒转导 和适应亲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 如何在功能上不同的位点上成核 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 支持 E 蛋白建立 T 谱系承诺调节状态。使用 条件双敲除，我们将解析所介导的 Bcl11b 基因调控效应的子集 通过该电路直接控制。