TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes.
TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes.
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DOI：
10.1371/journal.pgen.1010207
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发表时间：
2022-05
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影响因子：
4.5
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中科院分区：
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--
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在发育中的黑素细胞和黑色素瘤细胞中，激活增强子结合蛋白2转录因子家族(TFAP2)的多个类似基因参与了色素调节基因的表达，但它们与这些细胞的主要调节因子微眼炎转录因子(MITF)的相互作用尚不清楚。斑马鱼胚胎的单细胞序列分析表明，色素沉着基因只在表达tfap2的细胞亚群中表达，这一模型支持TFAP2促进MITF激活色素沉着基因表达的能力的模型。为了在SK-Mel-28黑色素瘤细胞中测试这一模型，我们删除了两个表达最高的TFAP2同源基因TFAP2a和Tfap2c，创建了TFAP2基因敲除(TFAP2-KO)细胞。然后，我们评估了基因表达、染色质可及性、TFAP2A和MITF的结合，以及染色质标记H3K27Ac和H3K27me3，它们分别是活性增强剂和沉默染色质的特征。对这些数据集的综合分析表明，TFAP2类似物直接激活富含色素和增殖作用的基因附近的增强子，并直接抑制富含细胞黏附作用的基因附近的增强子。一直以来，与WT细胞相比，TFAP2-KO细胞增殖较少，彼此黏附较多。TFAP2类似物和MITF协同激活增强子的一个子集，前者是MITF结合和染色质可及性所必需的。相比之下，TFAP2辅助物和MITF似乎不能协同抑制增强子。这些研究揭示了TFAP2深刻影响由MITF激活的一组基因，从而影响色素细胞和黑色素瘤细胞表型的机制。黑素细胞是皮肤中产生色素的细胞，其分化受基因调控网络的控制，该网络由主要调节蛋白MITF控制。值得注意的是，MITF活性的水平也决定了黑色素瘤细胞采用的侵袭性、增殖性或分化表型，这最终影响了患者的预后。TFAP2转录因子家族也调节黑素细胞分化和黑色素瘤表型。MITF和TFAP2如何合作调节这些细胞过程在很大程度上是未知的。在这里，我们利用基因组数据集的组合，证明了TFAP2同源基因1)冗余地促进斑马鱼胚胎黑素细胞的发育和加速色素沉着，2)可以保持开放或浓缩的染色质，3)通过促进MITF的活性调节其访问编码色素沉着和细胞增殖调节的基因，4)抑制细胞-细胞黏附基因的增强子。在黑色素瘤的细胞系模型中，TFAP2类似物的存在或不存在部分通过它们与MITF的相互作用来调节细胞增殖和细胞-细胞黏附之间的表型转换。
In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that TFAP2 facilitates MITF’s ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the former necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells. Differentiation of melanocytes, the pigment producing cells in skin, is controlled by a gene regulatory network governed by the master regulator protein, MITF. Remarkably, the level of MITF activity also determines the invasive, proliferative, or differentiated phenotype adopted by melanoma cells, which ultimately affects patient outcome. The TFAP2 family of transcription factors (TFAP2 paralogs) also regulate melanocyte differentiation and melanoma phenotype. How MITF and TFAP2 cooperate to regulate these cellular processes is largely unknown. Here, using a combination of genomic datasets, we show that TFAP2 paralogs 1) redundantly promote embryonic melanocyte development and accelerate pigmentation in zebrafish, 2) can maintain open or condensed chromatin, 3) regulate MITF activity by facilitating its access to genes encoding regulators of pigmentation and cell proliferation, 4) inhibit enhancers of cell-cell adhesion genes. In these ways, the presence or absence of TFAP2 paralogs mediates a phenotypic switch between cell proliferation and cell-cell adhesion in a cell line model of melanoma, in part through their interactions with MITF.
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