Regulation of the melanocyte lineage by the AP2 transcription factor family

描述（由申请人提供）：转录因子是控制所有类型前体细胞和干细胞分化的调控网络中的核心参与者。众所周知，转录因子调节基因表达，但它们如何调节分化尚不清楚。例如，在某些情况下，给定的转录因子在一种情况下促进细胞分化，而在另一种情况下阻止细胞分化。黑素细胞是探索细胞发育事件的理想模型细胞类型，因为它们的分化很容易被记录，它们不是生命所必需的，并且黑素细胞干细胞的丧失在哺乳动物中很容易通过随后的毛发变白而明显。此外，编码转录因子和其他调节分子的基因突变导致色素沉着发育障碍（如Hirschsprung病、Tietz综合征、Waardenburg综合征I- IV型、piebaldism）。我们的长期目标是了解黑素细胞谱系中发育事件的调控网络。在这里，我们重点关注转录因子激活蛋白2家族（TFAP2A-E）。TFAP2A似乎可以阻止黑素细胞干细胞的分化，因为TFAP2A的显性突变会导致Branchio Oculo Facial Syndrome (BOFS)，其特征是头发过早变白。相比之下，TFAP2A及其类似的TFAP2E似乎可以促进胚胎黑素细胞的分化，正如我们在斑马鱼中的研究所显示的那样。我们假设在黑素细胞前体中，TFAP2蛋白具有冗余功能，并与小眼相关转录因子（MITF）并行，MITF是黑素细胞生物学的中心调节因子，可激活影响黑素细胞分化的基因。为了测试该模型，我们将在斑马鱼中进行敲除/加回研究，在小鼠中产生适当的组织特异性双突变体，并在培养的人类黑素细胞中对TFAP2蛋白和MITF相互激活的基因启动子进行功能分析。接下来，我们将研究TFAP2A如何维持黑素细胞干细胞（MSC）的功能。我们假设在这种情况下，TFAP2A的关键功能是激活KIT。为了验证这一观点，我们将再次使用斑马鱼进行上浮试验，在神经冠特异性缺失TFAP2A的小鼠中检测头发变白，并评估与BOFS中头发变白相关的TFAP2A变体的功能特性。最后，我们将对黑色素细胞中TFAP2A表达的潜在调控因子进行系统分析，并对黑色素细胞中TFAP2A转录靶点进行全局分析，共同将TFAP2A定位到黑色素细胞中的调控网络中。我们的研究将揭示新的遗传和分子网络的基础上的正常和病理发展的黑素细胞。从这项工作中产生的新知识将特别在Waardenburg综合征和BOFS的病因学中具有价值。更一般地说，它将阐明在一个可处理的模型细胞类型分化的转录调控。

DESCRIPTION (provided by applicant): Transcription factors are central players in the regulatory networks that control differentiation in all types of precursor cells and stem cells. Itis well known that transcription factors regulate gene expression, but it is unclear how they regulate differentiation. For instance, in some cases a given transcription factor promotes cell differentiation in one context, and prevents it in another. Melanocytes are an ideal model cell type to explore cellular developmental events because their differentiation is easily scored, they are not essential for life, and loss of melanocyte stem cells in mammals is readily apparent by the consequent hair graying. Moreover, mutations in genes encoding transcription factors and other regulatory molecules cause developmental disorders of pigmentation (e.g., Hirschsprung's disease, Tietz syndrome, Waardenburg syndrome types I- IV, piebaldism). Our long-term objective is to understand the regulatory network governing developmental events in the melanocyte lineage. Here we focus on the transcription factor Activator Protein 2 family (TFAP2A-E). TFAP2A appears to prevent differentiation of melanocyte stem cells, because dominant mutations in TFAP2A cause Branchio Oculo Facial Syndrome (BOFS), which features premature hair graying. By contrast, TFAP2A and its paralog TFAP2E appear to promote differentiation of embryonic melanocytes, as shown by our work in zebrafish. We hypothesize that within melanocyte precursors, TFAP2 proteins function redundantly, and in parallel with microphthalmia-associated transcription factor (MITF), a central regulator of melanocyte biology, to activate genes that effect melanocyte differentiation. To test this model we will conduct knock-down/add-back studies in zebrafish, generate appropriate tissue- specific double mutants in mice, and conduct functional analysis of the promoter of a gene mutually activated by TFAP2 proteins and MITF in cultured in human melanocytes. Next we will examine how TFAP2A functions to maintain melanocyte stem cells (MSC). We hypothesize that in this context, the key function of TFAP2A is to activate KIT. To test this notion we will use zebrafish again for epistasis tests, examine hair graying in a mouse with neural-crest-specific deletion of TFAP2A, and assess functional properties of TFAP2A variants associated with hair graying in BOFS. Finally, we will conduct systematic analyses of potential regulators of TFAP2A expression in melanocytes, and a global analysis of TFAP2A transcriptional targets in melanocytes, which together will position TFAP2A into a regulatory network in melanocytes. Our studies will shed light on novel genetic and molecular networks that underlie normal and pathological development of melanocytes. The new knowledge generated from this work will be of value specifically in the etiology of Waardenburg syndrome and BOFS. More generally, it will illuminate the transcriptional regulation of differentiation in a tractable model cell type.