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

摘要 此申请是针对NOT-RM-19-009提交的。我们将使用共同基金的资源 从现有的数据库中扩大我们对潜在基因和调控相互作用的理解 黑素细胞生物学。我们父母资助的重点是黑素细胞干细胞(MSC)，这是一种易于处理的 其他成体干细胞的模型。特别是，我们感兴趣的是转录因子MITF和AP-2是如何 相互作用来调节黑素细胞的发育，在这项补充建议中，我们将利用表型 来自KOMP项目的信息，来自4D基因组的HIC数据，以及SNP/基因表达关联数据 从GTEx项目中更好地了解负责 黑素细胞的产生和维持。编码AP-2a的基因TFAP2A的突变导致Branchio- 眼面部综合征(BofS)，一种先天性缺陷，包括过早的头发白发。我们有 先前的研究表明，AP-2蛋白是黑素细胞形成所需的神经脊发育早期所必需的。 此外，AP-2蛋白可以与被认为是黑素细胞主要调节因子的MITF在基因上相互作用 血统。然而，AP-2和MITF在这个GRN中的相对位置，它们在 控制表达的基因组，以及这个网络中存在的基因的数量和类型保持不变 不清楚。我们最近确定GRN需要AP-2a和AP-2b的组合 在MSCs中控制增殖、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，包括早衰AS 见于BofS患者。我们现在正在通过执行scRNAseq和 SCATACseq实验揭示基因表达和顺式调控电路的变化 控制。此外，我们还进行了CUT&RUN，这是对CHIP-SEQ的改进，以鉴定基因组- MITF和TFAP2A在野生型、MITF缺失和TFAP2A缺失的黑色素瘤细胞系中的广泛结合位点， 随后在每个细胞系上执行RNA-SEQ。这些新数据提供了一个出色的平台 询问共同基金的数据集以了解系统生物学的组成和结构 管理MSC行为的GRN。简而言之，我们将整合所描述的表型和基因组数据 上图为：Komp关于色素缺陷小鼠的数据：来自两个黑色素瘤细胞系的HIC数据揭示了 相关的TFAP2增强子-启动子连接；以及将特定SNPs与基因表达联系起来的GTEx数据 级别。拟议的为期一年的项目的预期结果是加深对GRN的了解 控制骨髓间充质干细胞生长分化的平衡。对MITF机制的理解 以及TFAP2对MSC维护的贡献，以及这些因素是如何调节的，将具有广泛的 对衰老机制和黑色素瘤的发生和发展的影响，以及 揭示了这些公共数据集对系统级别分析的效用。

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.