The Role of Hox Transcription Factors in Cellular Plasticity

多细胞动物之所以复杂，是因为它们能够产生和维持由多种细胞类型组成的不同谱系，这些细胞类型共享几乎相同的基因组DNA。细胞命运由转录因子网络控制，转录因子网络在基因组染色质状态的背景下激活转录程序，实现给定谱系细胞的不同特性。然而，转录因子如何控制不同的细胞命运，在生物学上仍是一个悬而未决的问题。HOX转录因子代表了一个很好的模型来解决这一根本问题，因为它们活跃在沿双侧动物前后轴(AP)的所有谱系中。我们以Ubx为模型的初步数据表明，Hox转录因子除了在AP轴上指定区域身份的功能外，在谱系的发展和多样化中发挥着重要作用，其中一个机制是谱系特异性的多梳复合体招募或稳定到Ubx染色质部位，以抑制替代命运和早期谱系指定基因。我们的结果还表明，HOX编码的细胞和时间可塑性的限制对于在细胞的整个生命周期中稳定地维持细胞的命运是必要的。这些发现引发的一个基本问题是，在没有任何功能性HOX输入的情况下，已经承诺的谱系将如何发展，以及不含HOX的细胞是否比表达HOX的细胞更具可塑性。我们将通过分析无HOX的蜂窝环境如何影响谱系发育来解决这些问题。为此，我们将消除中胚层谱系中的HOX编码，并将在转录组水平上记录其影响。此外，我们还将在表型水平上表征该谱系的身份和形态。为了阐明HOX编码抑制多能性的机制基础，我们将识别那些对谱系稳定至关重要的基因组区域，因为它们在没有HOX转录因子的情况下转换为开放和可激活的状态。作为概念验证，我们将选择候选区域，并通过使用CRISPR/Cas9介导的基因组工程特定地删除这些区域来测试它们在体内通过谱系稳定的功能。最后，我们将比较不表达HOX的细胞和表达HOX的细胞的发育潜力，以阐明“去除”HOX编码的细胞是否更具可塑性，因此在受到刺激时更容易改变其身份。为此，我们将在对照和体内缺失HOX的中胚层细胞中错误表达另一种谱系的主调节因子，并将比较它们的表型和分子特性。总之，我们预计这项研究将对解决HOX编码的谱系稳定的机制基础至关重要，这可能被证明与重新编程策略高度相关。

Multicellular animals owe their complexity to their capacity to produce and maintain different lineages composed of multiple cell types that share virtually the same genomic DNA. Cell fates are controlled by networks of transcription factors (TFs) that act in the context of the genomic chromatin state to activate transcriptional programs realizing the distinct properties of cells of a given lineage. However, how TFs control different cell fates is still un unsolved question in biology. Hox TFs represent an excellent model to address this fundamental problem, since they are active in all lineages along the anterior-posterior (AP) axis of bilaterian animals. Our preliminary data using Ubx as a model show that Hox TFs, besides their well-described function in specifying regional identities along the AP axis, play a major role in the development and diversification of lineages, and that one of the mechanisms is the lineage-specific recruitment or stabilization of the Polycomb complex to Ubx chromatin sites for the repression of alternative fate and early lineage specification genes. Our results also imply that the Hox-encoded restriction of cellular and temporal plasticity is required for stably maintaining cell fates throughout the lifetime of a cell. One fundamental question arising from these findings is how an already committed lineage will develop in the absence of any functional Hox input and whether Hox-free cells are more plastic that Hox-expressing cells. We will tackle these questions by analyzing how a Hox-free cellular environment affects lineage development. To this end, we will eliminate the Hox code in the mesodermal lineage and will record the effects at the transcriptome level. In addition, we will characterize the identity and morphology of the lineage on the phenotypic level. To elucidate the mechanistic basis of the Hox-encoded suppression of multipotency, we will identify those genomic regions that are critical for lineage stabilization, as they are converted into an open and activatable status in the absence of Hox TFs. As a proof of concept, we will select candidate regions and test their function in lineage stabilization in vivo by lineage-specifically deleting these regions using CRISPR/Cas9 mediated genome engineering. And finally, we will compare the developmental potential of Hox-free and Hox-expressing cells to elucidate whether cells “stripped” of their Hox code are more plastic and can thus more easily change their identity when stimulated than cells expressing the inherent Hox code. To this end, we will mis-express a master-regulator of an alternative lineage in control and Hox-depleted mesodermal cells in vivo and will compare their phenotypic and molecular identities. In sum, we envision this study to be crucial in resolving the mechanistic basis of Hox-encoded lineage stabilization, which might prove to be highly relevant for reprogramming strategies.