An in-vivo screen of noncoding elements reveals that Daedalus is a gatekeeper of a novel Ikaros-dependent checkpoint during haematopoiesis I

The development of all lymphoid lineages relies on a tightly-controlled series of gene expression patterns as development proceeds through a series of progenitors1-8. These gene expression patterns are understood to be controlled by lineage-specifying transcription factors, however these factors often are found to have broad expression patterns and activities across lineages. Given their role in regulating cell-type specific gene expression, noncoding loci are thought be important in the lineage-specific regulation of this complex process6,9-16. To identify transcripts that might mark such loci, we performed transcriptomic analysis in terminally differentiated murine CD4 effector T lymphocytes. Our analysis identified a variety of loci containing dynamically expressed noncoding RNAs and we tested their role in the lymphoid lineage using an in-vivo CRISPR knockout-mouse screen. This approach revealed that a non-coding locus proximal to the haematopoietic transcription factor Ikaros17,18, named Daedalus, exerted control over the earliest fate decisions during lymphoid lineage commitment. Deletion of Daedalus led to a cell-autonomous reduction in bone marrow and thymic lymphoid progenitor populations, phenocopying Ikaros deficient strains. Strikingly, loss of Daedalus resulted in a loss of Ikaros protein expression without impacting total Ikaros transcript levels. Finally, we identify a novel Ikaros regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage specific manner. Daedalus appears to act as a gatekeeper of Ikaros’s broad lineage specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. To our knowledge, these findings represent the first illustration of how a transcription factor with broad lineage expression must work in concert with non-coding elements to orchestrate haematopoietic lineage specification and commitment. In order to uncover novel non-coding loci with the potential to regulate lymphoid cell biology, whole transcriptomic analysis of purified T-cell populations was conducted. Populations of CD4 “memory-like” (CD44hi CD62L-) and “naïve” (CD44- CD62Lhi) cells were sorted from mouse lymph nodes. Separately, naïve T-cells from wild-type mice were sorted and polarized under neutral “Th0” conditions, with stimulation from anti-CD28 and anti-CD3 antibodies, which bind the TCR and CD28 costimulatory receptor, stimulating antigen binding. In the “Th0” culture conditions, antibodies blocking Il-4 and interferon gamma (IFNγ) are also added to block Th1 and Th2 differentiation, and Il2 is added to stimulate T-cell differentiation and proliferation. In addition, purified populations of “naïve” CD4 T-cells were sorted from several reporter mouse lines were isolated, polarized into effector subsets in culture and sorted using reporter expression to generate highly purified effector populations. Naïve CD4 T-cells from IFNγ, Il-4, Il-9, Il-17 and FoxP3 reporter mice were sorted and polarized under Th1, Th2, Th9, Th17 and Treg conditions respectively. Purified populations were obtained by fluorescence-activated cell sorting of cells expressing the respective reporter genes to generate CD4 effector T-cell populations with a very high purity. RNA was extracted from these populations, along with from Th0-polarized effector CD4 cells and the naïve and memory populations described previously. This RNA was then used for a transcriptomic analysis by RNA-sequencing in order to identify differentially expressed RNAs between different effector T-cell populations that might mark important noncoding loci.

所有淋巴谱系的发育均依赖于一系列严格调控的基因表达模式，该过程需历经一系列祖细胞阶段1-8。已知此类基因表达模式由谱系特异性转录因子调控，但这些因子往往在不同谱系中展现出广泛的表达模式与活性。鉴于其在调控细胞类型特异性基因表达中的作用，非编码基因座被认为在这一复杂过程的谱系特异性调控中发挥重要作用6,9-16。为鉴定可标记此类基因座的转录本，我们对终末分化的小鼠CD4效应T淋巴细胞（CD4 effector T lymphocytes）开展了转录组分析（transcriptomic analysis）。本研究鉴定出一批包含动态表达非编码RNA的基因座，并通过体内CRISPR基因敲除小鼠筛选（in-vivo CRISPR knockout-mouse screen）验证了它们在淋巴谱系中的功能。该研究发现，位于造血转录因子Ikaros（haematopoietic transcription factor Ikaros）邻近区域的一个非编码基因座Daedalus，可调控淋巴谱系定型过程中的早期命运决定事件。敲除Daedalus会引发细胞自主性的骨髓与胸腺祖细胞群减少，其表型与Ikaros缺陷型小鼠品系一致。值得注意的是，Daedalus缺失会导致Ikaros蛋白表达水平下降，但不影响总Ikaros转录本水平。最后，我们鉴定出一个由Daedalus以淋巴谱系特异性方式调控的新型Ikaros依赖性红系-淋巴系检查点（erythroid-lymphoid checkpoint）。Daedalus似乎可作为Ikaros广谱谱系调控功能的守门基因，选择性稳定淋巴谱系中的Ikaros活性，并在其缺失时允许细胞向红系命运分化。据我们所知，本研究首次阐明了具有广谱谱系表达特性的转录因子需与非编码元件协同作用，以调控造血谱系的定型与特化。为挖掘具备调控淋巴细胞生物学潜能的新型非编码基因座，我们对纯化的T细胞群开展了全转录组分析。我们从小鼠淋巴结中分选得到CD4"记忆样"（CD44hi CD62L-）与"初始型"（CD44- CD62Lhi）细胞群。另外，我们从野生型小鼠中分选初始T细胞，并在中性"Th0"条件下进行极化培养：使用抗CD28与抗CD3抗体刺激，二者可结合T细胞受体（T cell receptor, TCR）与CD28共刺激受体，模拟抗原结合触发的信号通路。在Th0培养条件中，我们还添加了阻断白介素-4（Il-4）与干扰素γ（IFNγ）的抗体，以抑制Th1与Th2细胞分化，并添加Il-2以促进T细胞的分化与增殖。此外，我们从多个报告基因小鼠品系中分选纯化的"初始型"CD4 T细胞，在体外极化为效应细胞亚群，并通过报告基因表达分选出高纯度的效应细胞群。我们分别从干扰素γ、Il-4、Il-9、Il-17与FoxP3报告基因小鼠中分选初始CD4 T细胞，并分别在Th1、Th2、Th9、Th17与调节性T细胞（Treg）培养条件下进行极化。通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选出表达相应报告基因的细胞，以获得极高纯度的CD4效应T细胞群。我们从上述细胞群，以及Th0极化的效应CD4细胞、此前描述的初始型与记忆型细胞群中提取RNA。随后利用该RNA进行RNA测序（RNA-sequencing）转录组分析，以鉴定不同效应T细胞群之间差异表达的RNA，此类RNA或可标记重要的非编码基因座。