Dysregulated Plastin 3 (PLS3) levels associate with a wide range of skeletal and neuromuscular disorders and the most common types of solid and hematopoietic cancer. Most importantly, PLS3 overexpression protects against spinal muscular atrophy. Despite its crucial role in F-actin dynamics in healthy cells and its involvement in many diseases, the mechanisms that regulate PLS3 expression are unknown. Interestingly, PLS3 is an X-linked gene and all asymptomatic SMN1-deleted individuals in SMA-discordant families who exhibit PLS3 upregulation are female, suggesting that PLS3 may escape X chromosome inactivation. To elucidate mechanisms contributing to PLS3 regulation, we performed a multi-omics analysis in two SMA-discordant families using lymphoblastoid cell lines and iPSC-derived spinal motor neurons originated from fibroblasts. We show that PLS3 tissue-specifically escapes X-inactivation. PLS3 is located ∼500 kb proximal to the DXZ4 macrosatellite, which is essential for X chromosome inactivation. By applying molecular combing in a total of 25 lymphoblastoid cell lines (asymptomatic individuals, individuals with SMA, control subjects) with variable PLS3 expression, we found a significant correlation between the copy number of DXZ4 monomers and PLS3 levels. Additionally, we identified chromodomain helicase DNA binding protein 4 (CHD4) as an epigenetic transcriptional regulator of PLS3 and validated co-regulation of the two genes by siRNA-mediated knock-down and overexpression of CHD4. We show that CHD4 binds the PLS3 promoter by performing chromatin immunoprecipitation and that CHD4/NuRD activates the transcription of PLS3 by dual-luciferase promoter assays. Thus, we provide evidence for a multilevel epigenetic regulation of PLS3 that may help to understand the protective or disease-associated PLS3 dysregulation.Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved. PLS3调控失调与多种骨骼和神经肌肉疾病以及最常见的实体和造血恶性肿瘤相关。最重要的是，PLS3过表达可以保护脊髓肌萎缩不受影响。尽管PLS3在健康细胞的F-actin动力学中扮演着至关重要的角色并参与许多疾病，但其调控机制尚不清楚。有趣的是，PLS3是一种X连锁基因，在SMA不协调家族中，所有表现为PLS3上调的无症状SMN1缺失个体都是女性，这表明PLS3可能逃避X染色体失活。为了阐明有助于PLS3调控的机制，我们使用淋巴母细胞株和来源于成纤维细胞的iPSC衍生的脊髓运动神经元，在两个SMA不协调家族中进行了多组学分析。我们展示了PLS3组织特异性逃避X失活的情况。PLS3位于∼500 kb的DXZ4宏卫星旁边，该宏卫星对X染色体失活至关重要。通过对25个淋巴母细胞系（无症状个体，SMA个体和对照个体）进行分子梳理，我们发现DXZ4单体拷贝数与PLS3水平之间存在显著相关性。此外，我们确定染色质结构域螺旋框DNA结合蛋白4（CHD4）为PLS3的表观遗传转录调节因子，并通过siRNA介导的敲除和CHD4的过表达验证了两个基因的共同调节。我们展示了CHD4通过进行染色质免疫沉淀结合PLS3启动子，并通过双荧光素酶报告分析证明了CHD4 /NuRD活化PLS3的转录。因此，我们提供了一种多级表观遗传调控PLS3的证据，这有助于理解PLL3失调的保护性或疾病相关性。Copyright©2023年美国人类遗传学学会。Elsevier Inc.发表。版权所有。

Dysregulated Plastin 3 (PLS3) levels associate with a wide range of skeletal and neuromuscular disorders and the most common types of solid and hematopoietic cancer. Most importantly, PLS3 overexpression protects against spinal muscular atrophy. Despite its crucial role in F-actin dynamics in healthy cells and its involvement in many diseases, the mechanisms that regulate PLS3 expression are unknown. Interestingly, PLS3 is an X-linked gene and all asymptomatic SMN1-deleted individuals in SMA-discordant families who exhibit PLS3 upregulation are female, suggesting that PLS3 may escape X chromosome inactivation. To elucidate mechanisms contributing to PLS3 regulation, we performed a multi-omics analysis in two SMA-discordant families using lymphoblastoid cell lines and iPSC-derived spinal motor neurons originated from fibroblasts. We show that PLS3 tissue-specifically escapes X-inactivation. PLS3 is located ∼500 kb proximal to the DXZ4 macrosatellite, which is essential for X chromosome inactivation. By applying molecular combing in a total of 25 lymphoblastoid cell lines (asymptomatic individuals, individuals with SMA, control subjects) with variable PLS3 expression, we found a significant correlation between the copy number of DXZ4 monomers and PLS3 levels. Additionally, we identified chromodomain helicase DNA binding protein 4 (CHD4) as an epigenetic transcriptional regulator of PLS3 and validated co-regulation of the two genes by siRNA-mediated knock-down and overexpression of CHD4. We show that CHD4 binds the PLS3 promoter by performing chromatin immunoprecipitation and that CHD4/NuRD activates the transcription of PLS3 by dual-luciferase promoter assays. Thus, we provide evidence for a multilevel epigenetic regulation of PLS3 that may help to understand the protective or disease-associated PLS3 dysregulation.Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.