Role of the SAF-A SAP domain in X inactivation, transcription, splicing, and cell proliferation [RNA-seq]

SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase. To understand how SAF-A contributes to gene expression, mRNA splicing, and X chromosome inactivation we established a Auxin Inducible Degron cell line for SAF-A in RPE-1 cells. We then rescued SAF-A depletion using inducible point mutants on the SAP domain. We then performed total RNA-seq from WT and mutant cell lines at various time points after SAF-A depletion or allele replacement. We analyzed bulk differential gene expression, mRNA splicing, and allele-specific expression changes.

SAF-A在整个脊椎动物中均保守，并已被证实是调控多种核功能的重要因子，包括长链非编码RNA（lncRNA）定位、基因表达及mRNA剪接过程。SAF-A拥有多个功能结构域，其中包括一个可直接结合DNA的N端SAP结构域（SAP domain）。SAP结构域内丝氨酸S14与S26的磷酸化，对于有丝分裂过程中SAF-A的定位与功能至关重要，但目前尚不明确这些丝氨酸是否参与SAF-A的细胞间期功能。本研究探究了SAP结构域，以及SAP结构域丝氨酸S14和S26在X染色体失活、蛋白质动态调控、基因表达、剪接及细胞增殖中的作用。本研究结果显示，SAP结构域丝氨酸S14与S26是维持雌性细胞中XIST RNA（X染色体失活特异性RNA）定位，以及失活X染色体（Xi）上多梳蛋白依赖型组蛋白修饰所必需的。此外，本研究提供的证据表明，失活X染色体的定位信号存在于SAP结构域中。我们发现，SAP结构域并非维持基因表达所必需，且仅在mRNA剪接过程中发挥微弱作用。与之相反，SAF-A的SAP结构域，尤其是丝氨酸S14与S26，对于维持正常的蛋白质动态及正常细胞增殖是必需的。我们提出了一个模型：SAF-A丝氨酸S14与S26的动态磷酸化，介导了细胞间期内SAF-A与DNA相互作用的快速周转。为阐明SAF-A如何参与基因表达、mRNA剪接及X染色体失活，我们在RPE-1细胞中构建了SAF-A的生长素诱导型降解细胞系（Auxin Inducible Degron）。随后，我们通过SAP结构域上的诱导型点突变体挽救了SAF-A的敲除缺陷。之后，我们在SAF-A敲除或等位基因替换后的不同时间点，从野生型（WT）和突变体细胞系中提取总RNA并开展总RNA测序（total RNA-seq）。我们分析了整体差异基因表达、mRNA剪接及等位基因特异性表达的变化。