The role of Med1 in dental stem cell fate

 描述(申请人提供)：出生后细胞的命运由成体干细胞决定，成体干细胞存在于再生组织中，如外胚层附属物。虽然它们的分化能力已经被广泛证明，但指定细胞命运的机制仍然知之甚少。小鼠切牙为研究成体干细胞在再生组织中的命运提供了一个很好的模型系统。门牙干细胞在老鼠的整个生命周期中支持门牙的持续生长。缓慢分裂的牙齿上皮干细胞(DE-SC)驻留在颈环中，指定牙齿程序并再生牙齿上皮。干细胞的命运和再生主要由特定的转录程序控制。我们开发了一种独特的干细胞再生模型，在该模型中，外胚层附件(如牙齿和皮肤)的转录程序被转换。转录共激活因子中介复合体1(MED1)的基因组缺失导致釉质发育不全，DE-SCs无法启动正常牙齿上皮命运的转录程序。取而代之的是，DE-SCs建立了一个表皮程序，并在门牙中再生异位毛发，这与Sox2表达的延长和Notch信号的减少有关。鉴于这些惊人的初步结果，我们假设该介体调节控制牙齿干细胞细胞命运的转录因子。MED1的缺失改变了原有的牙齿命运转录程序，导致了一种新的表皮/毛发命运特异转录状态。对MED1缺失模型的进一步研究将使我们能够通过使用微阵列和芯片测序(Aim1)的全基因组分析来识别遗传MED1靶标，命运决定转录因子。我们预计，这些分析将揭示特定的增强子结构域，称为“超级增强子”，其中MED1被高度招募来组织命运特异的染色质状态。我们还打算确定这些MED1靶标和调控分子的功能，包括Sox2和Notch1/Hes1(AIM2)。我们将利用siRNA介导的基因沉默和在AIM 1中鉴定的MED1和MED1靶基因的过表达来确定它们在体外培养系统中通过谱系特异性标记监测它们在牙齿和表皮细胞命运中的作用。然后，我们将通过考虑牙齿间充质的贡献，确定它们在DE-SC/间充质三维共培养中的作用，类似于体内干细胞生态位系统。这项研究的成功完成有望揭示驱动牙齿细胞命运的特定转录程序，并更广泛地为外胚层组织的分化提供洞察力。

 DESCRIPTION (provided by applicant): Postnatal cell fates are determined by adult stem cells residing in regenerative tissues such as ectoderm appendages. While their differentiation capacity has been demonstrated extensively, mechanisms that specify cell fate remain poorly understood. Mouse incisor provides an excellent model system to study adult stem cell fate in a regenerating tissue. Incisor stem cells support the continuous growth of the incisors throughout the lifespan of the mouse. Slow dividing dental epithelial stem cells (DE-SC) residing in the cervical loop specify the dental program and regenerate the dental epithelia. The stem cell fate and regeneration are primarily controlled by a specific transcriptional program. We developed a unique stem cell regeneration model, in which the transcriptional program for ectoderm appendages such as tooth and skin is converted. Genomic deletion of one subunit of the transcriptional coactivator Mediator complex, Mediator 1 (Med1), resulted in enamel hypoplasia, in which DE-SCs fail to institute the transcriptional program for a normal dental epithelial fate. Instead, DE-SCs institute an epidermal program and regenerate ectopic hairs in the incisors associated with extended Sox2 expression and reduced Notch signaling. Given these striking preliminary results we hypothesize that the Mediator regulates the transcription factors that control the cell fate of dental stem cells. Med1 deletion alters the pre-existing transcription program for dental fate resulting in a new state of epidermal/hair fate specific transcription. Further study of the Med1 null model will allow us to identify genetic Med1 targets, fate determining transcription factors, through genome wide analyses using microarray and Chip-sequencing (Aim1). We anticipate that these analyses will reveal specific enhancer domains called "super-enhancers" in which Med1 is highly recruited to organize fate specific chromatin states. We also aim to determine the functionality of these Med1 targets and regulatory molecules including Sox2 and Notch1/Hes1 (Aim2). We will use siRNA mediated gene silencing and overexpression of Med1 and Med1 target genes identified in Aim 1 to determine their role in dental and epidermal cell fate as monitored by lineage specific markers in in vitro culture system. We will then determine their role in 3D co-culture of DE-SC/mesenchyme resembling in vivo stem cell niche system by considering the contribution of dental mesenchyme. Successful completion of this study is expected to uncover a specific transcription program that drives dental cell fate, and more broadly provide insight into the differentiation of ectodermal tissues.