Function and evolution of zinc finger transcription factors

与大多数转录因子不同，大的C2H2锌指家族在后生动物中表现出广泛的复制和多样化的选择。大多数C2H2蛋白的特性都很差，因此仍然不知道它们的功能是什么，以及是什么驱动了它们的进化轨迹。然而，在一个案例中，四足特异的KRAB-C2H2蛋白的表达，它代表了大约一半的人类C2H2蛋白。最近的证据表明，该家族通过招募KAP1辅阻遏子来进化来沉默逆转录元件。 我的实验室最近发现，C2H2蛋白的基因组结合位点高度多样化，并且每个人的KRAB-C2H2蛋白都与一类特定的内源性逆转录元件(ERE)结合。奇怪的是，大多数结合的ERE都是古老的，长期以来一直处于不活跃状态，这表明结合它们的蛋白质和/或ERE本身承担了额外的作用。我们还发现，包括KRAB-C2H2在内的C2H2蛋白通常具有独特的蛋白质-蛋白质相互作用(PPI)谱，这表明可以通过在相对较短的进化时间内改变效应器功能来获得新的宿主功能。这些观察表明了一种特定于基因组的调控网络模式，不同于进化发展文献中流行的保守调控模式，以及驱动基因扩张的不同力量。 在这里，我们提出了两条主要的实验路线来探索四足和非四足脊椎动物中的C2H2蛋白，以获得对它们的功能和进化的更广泛和更详细的看法。 (1)我们将绘制基因组结合位点图，以确定哺乳动物中同源KRAB-C2H2蛋白的DNA结合位点的变化速度，以及鱼类中最近进化的C2H2蛋白是否与ERE结合。 (2)探讨PPI与C2H2蛋白的相互作用机制及其进化。我们预计有三种不同的进化“模式”，每一种我们都将详细探索：(A)结构蛋白结合模块的多样化，(B)使用非结构或非结构域多肽作为新的PPI的模板，或(C)将C2H2(或其他结构域)用于PPI，这将是“兼职”的一个例子。 总体而言，该项目将对脊椎动物进化的研究以及我们对调控网络和元件的理解做出重大贡献。 **这是一份为期一年的探索奖助金的续签申请。在此期间，我的小组发表了两篇以前被描述为初步数据的论文，并提交了第三篇论文(见附录)。我感谢这笔资金，它允许生成新的初步数据，并期待在这项经过广泛修订(和重新命名)的申请的基础上有更长的资金间隔。

Unlike most transcription factors (TFs), the large C2H2 zinc finger family displays widespread duplication and diversifying selection in metazoans. Most C2H2 proteins are poorly characterized, and therefore it remains unknown what are their functional roles and what drives their evolutionary trajectories. In one case, however that of the tetrapod-specific KRAB-C2H2 proteins, which represent roughly half of all human C2H2 proteins recent evidence indicates that the family evolves to silence retroelements by recruiting the KAP1 corepressor. My lab recently showed that the genomic binding sites of C2H2 proteins are highly diverse and that each human KRAB-C2H2 protein binds a specific class of endogenous retroelements (EREs). Curiously, most of the EREs bound are ancient and have long been inactive, suggesting that the proteins that bind them, and/or the EREs themselves, have taken on additional roles. We have also found that C2H2 proteins, including KRAB-C2H2s, often have unique protein-protein interaction (PPI) profiles, suggesting that new host functions can be acquired by changing the effector function over relatively short evolutionary timescales. These observations suggest a genome-specific regulatory network model, distinct from the conserved regulatory models prevalent in the evolutionary development literature, as well as different forces that drive the gene expansions. Here, we propose two major lines of experimentation to explore C2H2 proteins in both tetrapod and non-tetrapod vertebrates, in order to gain a broader and more detailed view of their function and evolution. (1) We will map genomic binding sites to determine the rate at which DNA binding sites change for orthologous KRAB-C2H2 proteins across mammals, and whether EREs are bound at all by recently evolved C2H2 proteins in fish. (2) We will explore the mechanisms and evolution of PPIs with C2H2 proteins. We anticipate three different evolutionary “modes”, each of which we will explore in detail: (a) Diversification in structured protein-binding modules, (b) use of unstructured or non-domain polypeptide as a template for new PPIs, or (c) utilization of C2H2 (or other domains) for PPIs, which would represent an example of “moonlighting”. The project overall will make a major contribution to the study of vertebrate evolution, and to our understanding of regulatory networks and elements. **This is a renewal application for a Discovery Grant that was funded for one year. In the interim, my group has published two papers previously described as preliminary data, and submitted a third (see Appendices). I am grateful for the funding, which allowed generation of new preliminary data, and look forward to a longer funding interval on the basis of this extensively revised (and retitled) application.**