The role of Med1 in dental stem cell fate

 描述（由申请人提供）：出生后细胞命运由再生组织（如外胚层附件）中的成体干细胞决定。虽然它们的分化能力已被广泛证明，但对指定细胞命运的机制仍知之甚少。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。门牙干细胞支持小鼠整个寿命期间门牙的持续生长。位于宫颈环中的缓慢分裂的牙上皮干细胞（DE-SC）指定牙科程序并再生牙上皮。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，其中外胚层附属物如牙齿和皮肤的转录程序被转换。转录辅激活因子介体复合物的一个亚基的基因组缺失，介体1（Med 1），导致釉质发育不全，其中DE-SC未能制定正常牙齿上皮命运的转录程序。相反，DE-SCs建立了一个表皮程序，并在门牙中再生异位毛发，这与延长的Sox 2表达和减少的Notch信号有关。鉴于这些惊人的初步结果，我们假设，调解人调节的转录因子，控制牙齿干细胞的细胞命运。Med 1缺失改变了牙齿命运的预先存在的转录程序，导致表皮/毛发命运特异性转录的新状态。Med 1空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（Aim 1）的全基因组分析来鉴定遗传Med 1靶点，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Med 1是高度招募组织命运特异性染色质状态。我们还旨在确定这些Med 1靶标和调控分子（包括Sox 2和Notch 1/Hes 1（Aim 2））的功能。我们将使用siRNA介导的基因沉默和目标1中确定的Med 1和Med 1靶基因的过表达，以确定它们在体外培养系统中通过谱系特异性标记物监测的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，确定它们在类似于体内干细胞生态位系统的DE-SC/间充质的3D共培养中的作用。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，并更广泛地提供对外胚层组织分化的见解。

 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.