The role of PHD12 in epigenetic control of cranial neural crest EMT

描述(申请人提供)：神经脊(NC)是在脊椎动物胚胎中的其他衍生物中，在背部神经管中产生的一种瞬时胚胎多能细胞群，用于形成头面部骨骼的软骨和骨。因此，颅面畸形通常归因于神经脊细胞发育的问题，根据神经脊细胞发育受阻的不同阶段，可能会表现出截然不同的颅面异常。上皮向间充质转化(EMT)是真正的神经脊程序启动前的第一个事件，这一步骤的中断可导致非常严重的颅面畸形。EMT伴随着钙粘素家族成员表达的变化，包括NC分层前Cad6B的下调。这一过程受Snail家族成员的调控，Snail家族成员直接结合Cad6B启动子并抑制其转录。然而，抑制和激活信号在EMT中的作用机制可能是复杂的，涉及多个相互关联的因素。在这里，我们探索表观遗传修饰在这一过程中的可能作用，特别是PHD12(命名为与植物同源域12的同源基因)的作用，PHD12是与Snail2合作在神经脊诱导过程中发现上调的基因。不同的研究表明，Snail和PHD12都可以募集Sin3A/HDAC复合体。这个大的多蛋白复合体能够使位于启动子附近的组蛋白去乙酰化，从而抑制目标基因。虽然Snail2可以与Cad6B的E盒结合，但我们假设它需要一个能够阅读表观遗传标记并招募抑制复合体Sin3A的伴侣。本申请的目的是证明Snail2和PHD12之间的直接相互作用使招募抑制复合体Sin3A/HDAC通过组蛋白去乙酰化完成Cad6B的表达。本申请涉及三个具体目标，包括：目标1，表征PHD12在NC EMT中的作用。我们的初步数据显示，PHD12在适当的时间和位置表达，以便在NC分层之前与NC相关联。我们假设并将测试PHD12的存在是否影响NC EMT相关基因。在具体目标2中，我们将确定Snail2、PHD12和Sin3A相互作用的机制分析，以及它们在Cad6B抑制中的作用。我们的初步芯片实验提供了PHD12与Cad6B基因座相互作用的证据。我们假设并将测试Snail2-PHD12-Sin3A之间的相互作用，以及这种相互作用是否需要与Cad6B基因位点结合，以通过启动子去乙酰化来抑制它。最后，在特定的目标3中，将确定PHD12在NC细胞迁移和颅面衍生物形成中的作用。我们将集中于PHD12在神经峰向颧弓迁移和随后分化的长期影响中所起的作用。为了将这一点延伸到神经脊细胞形成关键的颅面衍生物的以后阶段，将允许鸡胚胎在卵子中发育到后期(例如颧弓阶段；软骨和骨形成；腭架形成)，以测试早期击倒是否会导致以后面部衍生物的迁移和/或形成方面的问题。综上所述，我们将系统地讨论PHD12在NC EMT中的表观遗传学作用以及它对颅面衍生品形成的影响。

DESCRIPTION (provided by applicant): Neural crest (NC) is a transient embryonic multipotent group of cells that arises within the dorsal neural tube, to forms cartilage and bone of the craniofacial skeleton, among other derivatives in vertebrate embryos. Therefore, craniofacial abnormalities are usually attributed to problems in neural crest cell development and depending of which phase of neural crest cell development is disrupted, very different craniofacial anomalies can manifest. Epithelial-to-mesenchymal transition (EMT) is one of the first events before initiation of the bona fide neural crest program, and the disruption of this step can result in very severe craniofacial anomalies. EMT is accompanied by changes in expression of members of the cadherin family molecules, including the down-regulation of Cad6B prior NC delamination. This process is regulated by Snail family members, which directly bind the Cad6B promoter and repress its transcription. However, the mechanisms underlying the role of repressive and activating signals in EMT are likely to be complex and involve multiple and interconnected factors. Here, we explore the possible role of epigenetic modification in this process and specifically the role of PHD12 (named for homology to plant homeodomain 12), a gene discovered as upregulated during neural crest induction, in cooperation with Snail2. Different studies have demonstrated that both Snail and PHD12 can recruit Sin3A/HDAC complex. This large multiprotein complex is able to deacetylate the histones located on the proximity of promoters to repress the target gene. Although Snail2 can bind to the E-boxes of Cad6B, we hypothesize it requires a partner able to read the epigenetic marks and recruit the repressive complex Sin3A. The goal of this application is to demonstrate that the direct interaction between Snail2 and PHD12 makes possible to recruit the repressive complex Sin3A/HDAC to complete shutdown Cad6B expression via histone deacetylation. This application involves three specific aims that include: Aim 1, characterize the role of PHD12 in NC EMT. Our preliminary data show that PHD12 is expressed in a time and location appropriate to be associated with NC prior their delamination. We hypothesize and will test whether the presence of PHD12 affects the NC EMT related genes. In the Specific Aim 2, we will determine the mechanistic analysis of Snail2, PHD12 and Sin3A interaction, and their role in Cad6B repression. Our preliminary ChIP experiments provide evidence that PHD12 interacts with Cad6B locus. We hypothesize and will test the interaction between Snail2-PHD12-Sin3A as well as the requirement of this interaction to bind to the Cad6B locus to repress it via promoter deacetylation. Finally, in the Specific Aim 3 will determine the role of PHD12 in the migration of NC cells and the formation of craniofacial derivatives. We will focus on the role of PHD12 in long term effects on neural crest migration to the branchial arches and subsequent differentiation. To extend this to later times when neural crest cells are forming critical craniofacial derivatives, chick embryos will be allowed to develop in ovo to later stages (e.g. branchial arch stages; cartilage and bone formation; palatal shelf formation) to test if early knockdown leads to later problems in migration and/or formation of facial derivatives. Taking together we will systematically address the epigenetic role of PHD12 on NC EMT and it implication on the formation of craniofacial derivatives.