Cornell- Common Fund Data Supplement Regulation of the Melanocyte Lineage by the AP2 Transcription Factor Family

摘要 本申请是根据NOT-RM-19-009提交的。我们将利用共同基金资源 从现有的数据库，以扩大我们对基因和调控相互作用的理解， 黑素细胞生物学我们的母基金的重点是黑色素细胞干细胞（MSC），这是一个易于处理的 其他成体干细胞的模型。具体来说，我们感兴趣的是转录因子MITF和AP-2如何在转录过程中发挥作用。 相互作用以调节黑素细胞发育，在本补充建议中，我们将利用表型 来自KOMP项目的信息、来自4D核组的HiC数据和SNP/基因表达关联数据 从GTex项目获得更好的了解基因调控网络（GRNs）负责 黑素细胞的生成和维持。TFAP 2A（编码AP-2a的基因）的突变导致鳃裂症。 眼面部综合征（BOFS），一种先天性缺陷，包括过早的头发变白。我们有 先前显示AP-2蛋白在神经嵴发育早期需要用于黑素细胞形成。 此外，AP-2蛋白可以与MITF基因相互作用，MITF被认为是黑素细胞的主要调节因子 脉然而，AP-2和MITF在该GRN内的相对位置，它们如何在 控制表达的基因组，以及存在于这个网络中的基因的数量和类型， 不清楚我们最近确定，GRN需要AP-2a和AP-2b的组合 调节MSC的增殖、分化和存活之间的平衡， 突变等位基因的组合导致不同的色素沉着表型，包括过早变灰， 在BOFS患者中观察到。我们现在正在研究这些变化是如何发生的， scATACseq实验，以揭示基因表达和顺式调节回路的改变， 对照此外，我们还进行了CUT&RUN，这是对ChIP-seq的改进，以识别基因组- 野生型、MITF缺失和TFAP 2A缺失的黑素瘤细胞系中MITF和TFAP 2A的广泛结合位点， 随后对每个细胞系进行RNA-seq。这些新数据提供了一个出色的平台， 询问共同基金数据集，以了解系统生物学的组成和结构， GRN管理MSC行为。简而言之，我们将整合表型和基因组数据描述 以上是：关于色素沉着缺陷小鼠的KOMP数据：来自两种黑色素瘤细胞系的HiC数据， 相关的TFAP 2增强子-启动子连接;以及将特定SNP与基因表达联系起来的GTex数据 程度.拟议的一年期项目的预期成果是更深入地了解GRN 控制MSC生长和分化的平衡。了解MITF的机制 和TFAP 2有助于MSC的维护，以及这些因素是如何调节的，将有一个广泛的 关于衰老机制和黑色素瘤的发生和进展的影响，以及 揭示了这些公共数据集用于系统级分析的实用性。

Abstract This application is being submitted in response to NOT-RM-19-009. We will use Common Fund resources from available databases to broaden our understanding of the genes and regulatory interactions underlying melanocyte biology. The focus of our parent grant is the melanocyte stem cell (MSC), which is a tractable model of other adult stem cells. Specifically, we are interested in how the transcription factors MITF and AP-2 interact to regulate melanocyte development, and in this supplement proposal we will utilize phenotype information from KOMP project, HiC data from the 4Dnucleome, and SNP/gene expression association data from the GTex project to gain greater understanding of the gene regulatory networks (GRNs) responsible for melanocyte generation and maintenance. Mutations in TFAP2A, the gene encoding AP-2a, cause Branchio- Oculo-Facial syndrome (BOFS), a congenital defect which includes premature hair graying. We have previously shown that AP-2 proteins are required early in neural crest development for melanocyte formation. Further, AP-2 proteins can interact genetically with MITF, considered the master regulator of the melanocyte lineage. However, the relative positions of AP-2 and MITF within this GRN, how they function at the level of the genome to control expression, and the numbers and types of genes present within this network remain unclear. We have recently determined that a combination of AP-2a and AP-2b are required for the GRN governing the balance between proliferation, differentiation, and survival in MSCs and that different combinations of mutant alleles leads to different pigmentation phenotypes, including premature graying as seen in BOFS patients. We are now examining how these changes occur by performing scRNAseq and scATACseq experiments to reveal alterations in gene expression and cis-regulatory circuitry compared with controls. Further, we have carried out CUT&RUN, an improvement over ChIP-seq, to identify the genome- wide binding sites of MITF and TFAP2A in wild-type, MITF-deleted, and TFAP2A-deleted melanoma cell lines, subsequently performing RNA-seq on each cell line. These new data provide an outstanding platform to interrogate Common Fund datasets for a systems biology understanding of the components and structure of the GRN governing MSC behavior. In brief, we will integrate the phenotype and genomic data described above with: KOMP data on mice with pigmentation defects: HiC data from two melanoma cell lines revealing relevant TFAP2 enhancer-promoter connections; and GTex data to link specific SNPs with gene expression levels. The expected outcome of the proposed one-year project is a deeper understanding of the GRN controlling the balance of MSC growth and differentiation. An understanding of the mechanisms whereby MITF and TFAP2 contribute to MSC maintenance, alongside how these factors are regulated, will have a broad impact regarding the mechanisms of aging and the initiation and progression of melanoma, as well as revealing the utility of these public datasets for systems level analysis.