Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2023 Jun;299(6):104713. doi: 10.1016/j.jbc.2023.104713. Epub 2023 Apr 13.
Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template-driven manner in the endoplasmic reticulum and Golgi through the activity of a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wild-type cells. Additionally, RNA sequencing revealed that TFCP2 regulates the expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and the knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth.
硫酸乙酰肝素 (HS) 是一种长而线性的多糖，广泛表达于所有动物细胞中，在许多细胞过程中发挥关键作用，包括细胞信号转导和发育。HS 组装的失调与生理病理条件有关，如肿瘤发生和罕见的遗传疾病。HS 的生物合成在非模板驱动的方式下发生在内质网和高尔基体中，通过一大组生物合成酶的活性。虽然人们对其生物合成有了很多了解，但对不同组织类型和疾病状态下 HS 组装的调节知之甚少。为了解决这一知识空白，我们最近进行了全基因组 CRISPR/Cas9 筛选，以鉴定 HS 生物合成的新调节因子。在这些筛选中，我们确定了α珠蛋白转录因子 TFCP2 是一个重要的候选因子。为了研究 TFCP2 在 HS 组装中的作用，我们使用 CRISPR/Cas9 系统靶向人黑色素瘤细胞中的 TFCP2 表达。TFCP2 敲除细胞表现出与细胞表面 HS 结合的成纤维细胞生长因子减少、HS 组成改变以及与野生型细胞相比生长速度减慢。此外，RNA 测序显示 TFCP2 调节多种参与 HS 组装的酶的表达，包括分泌型内磺肽酶 SULF1。TFCP2 活性的药理学靶向同样减少了生长因子的结合并增加了 SULF1 的表达，并且在 TFCP2 突变细胞中敲低 SULF1 的表达恢复了黑色素瘤细胞的生长。总之，这些研究确定了 TFCP2 是 HS 的一种新型转录调节因子，并强调了 HS-蛋白相互作用可能是减缓黑色素瘤生长的一个潜在靶点。
