Moore Stephen P G, Ganesh Krishnan Shripushkar, Jaswanth Kothari Rutu, Prince Noah B, Kenny Colin, Zhang Chao, Lang Deborah
Department of Dermatology, Boston University, Boston, MA 02118, USA.
Bioinformatics Program, Boston University, Boston, MA 02118, USA.
Genes (Basel). 2025 May 14;16(5):577. doi: 10.3390/genes16050577.
BACKGROUND/OBJECTIVES: PAX3 is a transcription factor that drives melanoma progression by promoting cell growth, migration, and survival, while inhibiting cellular terminal differentiation. However, known PAX3 target genes are limited and cannot fully explain the wide impact of PAX3 function. The PAX3 protein can regulate DNA through two separate binding domains, the Paired Domain (PD) and Homeodomain (HD), which bind different DNA motifs. It is not clear if these two domains bind and work together to regulate genes and if they promote all or only a subset of downstream cellular events.
PAX3 direct downstream targets were identified using Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assays in SK-MEL-5 melanoma cells. PAX3-binding genomic regions were identified through MACS2 peak calling, and peaks were categorized based on the presence of PD and/or HD binding sites (or neither) through HOMER motif analysis. The peaks were further characterized as Active, Primed, Poised, Repressed, or Closed based on ATAC-seq data and CUT&RUN for histone Post-Translational Modifications H3K4me1, H3K4me3, H3K27me3, and H3K27Ac.
This analysis revealed that most of the PAX3 binding sites in the SK-MEL-5 cell line were primarily through the PD and connected to Active genes. Surprisingly, PAX3 does not commonly act as a repressor in SK-MEL-5 cells. Pathway analysis identified genes involved with transcription, RNA modification, and cell growth. Peaks located in distal enhancer elements were connected to genes involved in neuronal growth, function, and signaling.
Our results reveal novel PAX3 regulatory regions and putative genes in a melanoma cell line, with a predominance of PAX3 PD binding on active sites.
背景/目的：PAX3是一种转录因子，通过促进细胞生长、迁移和存活，同时抑制细胞终末分化来驱动黑色素瘤进展。然而，已知的PAX3靶基因有限，无法完全解释PAX3功能的广泛影响。PAX3蛋白可通过两个独立的结合域——配对结构域（PD）和同源结构域（HD）来调节DNA，这两个结构域结合不同的DNA基序。目前尚不清楚这两个结构域是否结合并共同作用来调节基因，以及它们是促进所有下游细胞事件还是仅促进一部分。
在SK-MEL-5黑色素瘤细胞中使用核酸酶靶向切割与释放（CUT&RUN）分析来鉴定PAX3直接下游靶点。通过MACS2峰值检测确定PAX3结合的基因组区域，并通过HOMER基序分析根据PD和/或HD结合位点（或两者都无）的存在对峰值进行分类。根据ATAC-seq数据以及组蛋白翻译后修饰H3K4me1、H3K4me3、H3K27me3和H3K27Ac的CUT&RUN结果，将这些峰值进一步表征为活跃、准备就绪、平衡、抑制或封闭。
该分析表明，SK-MEL-5细胞系中大多数PAX3结合位点主要通过PD，并与活跃基因相关。令人惊讶的是，PAX3在SK-MEL-5细胞中通常不作为抑制因子起作用。通路分析确定了与转录、RNA修饰和细胞生长相关的基因。位于远端增强子元件中的峰值与参与神经元生长、功能和信号传导的基因相关。
我们的结果揭示了黑色素瘤细胞系中新型的PAX3调控区域和推定基因，PAX3的PD在活跃位点上的结合占主导地位。
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