Regulation of the Melanocyte Lineage by the AP2 Transcription Factor Family

AP2 转录因子家族对黑素细胞谱系的调节

• 批准号: 10607024
• 负责人: Robert Aaron Cornell
• 金额: $ 54.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-12 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10607024
• 关键词: ATAC-seq; Acceleration; Address; Aging; Apoptosis; Binding; Biological Assay; Branchio-Oculo-Facial Syndromes; Cell Aging; Cell Differentiation process; Cell Line; Cell Maintenance; Cells; ChIP-seq; Chromatin; Data; Defect; Development; Differentiation and Growth; Embryo; Enhancers; Enterobacteria phage P1 Cre recombinase; Epithelial; Equilibrium; Exhibits; Family; Feedback; Gene Activation; Gene Expression; Genes; Genetic Epistasis; Genetic Transcription; Growth; Hair; Hair Removal; Hair follicle structure; Hair shaft structure; Homeostasis; Human; Improve Access; In Vitro; Ligase; MEL Gene; Maintenance; Malignant Neoplasms; Mediating; Melanoma Cell; Modeling; Mus; Mutation; Natural regeneration; Neural Crest; Normal Cell; Outcome; Pathway interactions; Patients; Pigments; Proliferating; Proteins; Regulation; Regulatory Element; Regulatory Pathway; Reporter; Research; Role; Signal Transduction; Skin; Stem Cell Development; Study models; Sumoylation Pathway; Syndrome; TFAP2A gene; Testing; Tissues; Transactivation; Transcription Coactivator; Transcription Factor AP-2 Alpha; Variant; Zebrafish; activating transcription factor; adult stem cell; base; cell behavior; cell type; enhancer-binding protein AP-2; experimental study; gene regulatory network; in vitro Assay; in vivo; inhibitor; melanoblast; melanocyte; melanoma; member; microphthalmia-associated transcription factor; mutant; overexpression; paralogous gene; pluripotency; premature; prevent; recruit; regenerative; senescence; stem cell function; stem cell niche; stem cells; transcription factor; transcriptome sequencing

Defects in the maintenance of stem cells can result in loss of the ability of tissues to regenerate or, conversely, in dysregulated growth and cancer. How the stem cell resists losing its regenerative qualities while retaining control of proliferation remains poorly understood. The melanocyte stem cell (MSC) is an ideal model for the study of adult stem cells in general. Either the apoptosis or differentiation of this cell type can cause hair graying, and mutations in MITF, which encodes the master regulator of melanocyte development, have been shown to cause hair graying in mice. Mutations in TFAP2A (Transcription Factor Activating-enhancer binding Protein 2 alpha) cause branchio-oculo-facial syndrome (BOFS), which includes premature hair graying. However, the mechanisms whereby TFAP2 proteins contribute to the relevant regulatory networks in MSCs remain unclear. Based on extensive preliminary data from zebrafish, mice, and cultured human melanocytes, our overall hypothesis is that AP-2 activity regulates pluripotency, growth and differentiation in melanocytes and MSCs, working in parallel with MITF and regulated by KCTD15-mediated sumoylation. In Aim 1 we will test the hypothesis that that cell-autonomous TFAP2 activity, provided redundantly by two paralogs, is necessary for the maintenance of MSCs, and that BOFS-associated TFAP2A variants inhibit both paralogs, leading to ectopic differentiation of MSCs and to hair graying. We will test this hypothesis in mice by deleting the relevant Tfap2 paralogs in MSCs or in cells of the MSC niche, and examining hair graying and MSC status. In Aim 2 we will test the hypothesis that TFAP2 paralogs serve as pioneer factors at a subset of the loci to which MITF binds, facilitating MITF-mediated activation of genes that promote growth and differentiation. This will entail deleting TFAP2 paralogs from a melanoma cell line and then assessing open chromatin using ATAC- SEQ, and binding of MITF using ChIP-SEQ. We will carry out parallel experiments in primary melanoblasts isolated from zebrafish embryos. In Aim 3 we will test the hypothesis that a negative feedback loop in TFAP2A signaling is necessary for the correct balance of maintenance and recruitment of MSCs. We found that a potent inhibitor of TFAP2-paralog transactivation activity, KCTD15, is expressed in melanocytes, and that zebrafish kctd15a/b mutants exhibit supernumerary, ectopic melanocytes. We will test the model that Kctd15- mediated sumoylation of Tfap2a blocks its transactivation activity, testing sumoylation in vitro. We will test kctd15a for a role in controlling melanocyte differentiation by abrogating its expression in the melanocyte lineage. We will also test the prediction that inhibition of TFAP2 mediates the effects of KCTD15 in the melanocyte lineage. The expected outcome of the proposed research is a deeper understanding of pathways that regulate the balance of growth and differentiation of MSCs. An understanding of the mechanisms whereby MITF and TFAP2 proteins contribute to MSC maintenance, and of how these factors are regulated, will have a broad impact as they participate in mechanisms of aging and in the initiation and progression of melanoma.

干细胞维护缺陷可能导致组织再生能力丧失，或者相反， 生长失调和癌症。干细胞如何在保留再生能力的同时抵抗丧失再生能力 对扩散的控制仍知之甚少。黑素细胞干细胞（MSC）是一个理想的模型 成体干细胞的一般研究。这种细胞类型的凋亡或分化都会导致毛发 头发变白和 MITF 突变（编码黑色素细胞发育的主调节因子）已被证实 已被证明会导致小鼠头发变白。 TFAP2A（转录因子激活增强子结合）突变 蛋白质 2α) 会导致鳃眼面部综合征 (BOFS)，其中包括头发过早变白。 然而，TFAP2 蛋白对 MSC 中相关调控网络的贡献机制 仍不清楚。基于来自斑马鱼、小鼠和培养的人类黑素细胞的大量初步数据， 我们的总体假设是 AP-2 活性调节黑素细胞的多能性、生长和分化 和 MSC，与 MITF 并行工作并受 KCTD15 介导的 sumoylation 调节。在目标 1 中，我们将 检验以下假设：由两个旁系同源物冗余提供的细胞自主 TFAP2 活性是 维持 MSC 所必需的，并且 BOFS 相关的 TFAP2A 变体抑制两种旁系同源物， 导致间充质干细胞异位分化和头发变白。我们将通过删除小鼠来测试这个假设 MSC 或 MSC 生态位细胞中的相关 Tfap2 旁系同源物，并检查头发变白和 MSC 状态。 在目标 2 中，我们将测试 TFAP2 旁系同源物作为基因座子集的先锋因子的假设 MITF 结合，促进 MITF 介导的基因激活，从而促进生长和分化。这 将需要从黑色素瘤细胞系中删除 TFAP2 旁系同源物，然后使用 ATAC 评估开放染色质 SEQ，以及使用 ChIP-SEQ 结合 MITF。我们将在原代成黑细胞中进行平行实验 从斑马鱼胚胎中分离出来。在目标 3 中，我们将测试 TFAP2A 中的负反馈循环这一假设 信号传导对于间充质干细胞的维持和招募的正确平衡是必要的。我们发现一个 TFAP2-paralog 反式激活活性的有效抑制剂 KCTD15 在黑素细胞中表达，并且 斑马鱼 kctd15a/b 突变体表现出多余的异位黑素细胞。我们将测试Kctd15-的模型 Tfap2a 介导的 sumoylation 阻断其反式激活活性，在体外测试 sumoylation。我们将测试 kctd15a 通过消除其在黑素细胞中的表达来控制黑素细胞分化 血统。我们还将测试抑制 TFAP2 介导 KCTD15 在 黑素细胞谱系。拟议研究的预期结果是对途径有更深入的了解 调节 MSC 生长和分化的平衡。了解其中的机制 MITF 和 TFAP2 蛋白有助于 MSC 的维持，以及这些因素如何调节，将会有一个 由于它们参与衰老机制以及黑色素瘤的发生和进展，因此具有广泛的影响。

项目成果

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells.

• DOI: 10.7554/elife.06857
• 发表时间: 2015-03-24
• 期刊: eLife
• 影响因子: 7.7
• 作者: Laurette P;Strub T;Koludrovic D;Keime C;Le Gras S;Seberg H;Van Otterloo E;Imrichova H;Siddaway R;Aerts S;Cornell RA;Mengus G;Davidson I
• 通讯作者: Davidson I

Motility phenotype in a zebrafish vmat2 mutant.

• DOI: 10.1371/journal.pone.0259753
• 发表时间: 2022
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Sveinsdóttir HS;Decker A;Christensen C;Lucena PB;Þorsteinsson H;Richert E;Maier VH;Cornell R;Karlsson KÆ
• 通讯作者: Karlsson KÆ

MITF reprograms the extracellular matrix and focal adhesion in melanoma.

MITF在黑色素瘤中重新编程细胞外基质和局灶性粘附。

• DOI: 10.7554/elife.63093
• 发表时间: 2021-01-13
• 期刊: eLife
• 影响因子: 7.7
• 作者: Dilshat R;Fock V;Kenny C;Gerritsen I;Lasseur RMJ;Travnickova J;Eichhoff OM;Cerny P;Möller K;Sigurbjörnsdóttir S;Kirty K;Einarsdottir BÓ;Cheng PF;Levesque M;Cornell RA;Patton EE;Larue L;de Tayrac M;Magnúsdóttir E;Ögmundsdóttir MH;Steingrimsson E
• 通讯作者: Steingrimsson E

TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes.

• DOI: 10.1371/journal.pgen.1010207
• 发表时间: 2022-05
• 期刊: PLoS genetics
• 影响因子: 4.5

BRN2 is a non-canonical melanoma tumor-suppressor.

• DOI: 10.1038/s41467-021-23973-5
• 发表时间: 2021-06-17
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Hamm M;Sohier P;Petit V;Raymond JH;Delmas V;Le Coz M;Gesbert F;Kenny C;Aktary Z;Pouteaux M;Rambow F;Sarasin A;Charoenchon N;Bellacosa A;Sanchez-Del-Campo L;Mosteo L;Lauss M;Meijer D;Steingrimsson E;Jönsson GB;Cornell RA;Davidson I;Goding CR;Larue L
• 通讯作者: Larue L