FUNCTIONAL GENOMICS AND MECHANISM OF BCL11B ACTION IN LYMPHOCYTE COMMITMENT

摘要/项目总结 Bcl 11 b是一种双功能锌指转录因子，对TCRαβ T的谱系定型至关重要 细胞Bcl 11b基因在细胞经历发育转变时被精确地激活， 它维持αβ T细胞身份和此后多个T细胞亚群的功能。基因，它 在最初的T细胞定型过程中保持沉默，包括所有先天淋巴细胞（ILC）和自然淋巴细胞（NK）的关键调节因子。 杀伤细胞（NK）。其中一些基因在成熟T细胞中也被Bcl 11b持续抑制。然而，在这方面， 令人惊讶的是，Bcl 11b也在一类特殊的先天淋巴样细胞ILC 2细胞中表达。ILC 2细胞还 需要Bcl 11b作为其身份，但同时，它们表达Bcl 11b沿着Id 2和其他基因， Bcl 11b通常在T细胞中沉默。一个主要的问题是Bcl 11b在ILC 2和早期的作用是如何如此不同的 T细胞（pro-T细胞）。我们建议使用功能基因组学和基因工程来确定这是如何工作的。 网络解剖，以阐明造成差异的机制。 我们来自ChIP-seq和蛋白质组学分析的初步证据表明，Bcl 11b仅与不同的 在两种细胞环境中部分重叠的位点组，并与不同的转录伴侣相互作用; 这与调节几乎完全不重叠的Bcl-11b敏感基因组有关， 两个上下文。为了解释这些差异，我们首先区分功能性位点和可能非功能性位点 其中Bcl 11b结合DNA，通过鉴定其对与其组装的辅助调节因子复合物的影响。因此， 定位了Bcl 11b对染色质调节具有局部不可或缺的成核作用的位点子集 辅因子和/或其他特定的相互作用伙伴。其次，我们计划绘制基因组区域， 染色质成环相互作用和区室边界也可以显示依赖于Bcl 11b表达。 这些结果将确定Bcl 11b在每个细胞环境中的结合位点，其中该结合是分子上的。 不降低为了确定这些差异的根本原因，我们将测试Bcl 11b自身的位点结合 偏好可以通过其它转录因子的存在来建立。我们至少发现了 另外两种重要的序列特异性转录因子，例如，（1）在许多情况下与Bcl 11b广泛相互作用， 其结合位点在T细胞背景下，但（2）在ILC 2细胞中代表不足。利用逆转录病毒转导 和CRISPR技术适用于pro-T细胞，我们将引入或删除假定的相互作用伴侣， 在ILC 2和pro-T细胞中，关键性地评估它们在引导结合和 Bcl 11b的功能我们将应用这种方法来解释Bcl 11b如何在细胞中的不同功能位点成核。 pro-T细胞和ILC 2。最后，一个特别重要的相互作用是基因网络电路， 我们已经证明Bcl 11b在建立T-谱系定型调节状态中支持E蛋白。使用 条件性双敲除，我们将解决介导的Bcl 11b基因调控效应的子集， 通过这个电路从那些被直接控制的。

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.