Zhang Erchen, Peng Lei, Yuan Kejia, Ding Zexian, Yi Qi
The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University Health Science Center, 410013 Changsha, Hunan, China.
Central South University Institute of Reproduction and Stem Cell Engineering, 410013 Changsha, Hunan, China.
Front Biosci (Landmark Ed). 2025 Jan 21;30(1):26426. doi: 10.31083/FBL26426.
α thalassemia/mental retardation syndrome X-linked (ATRX) serves as a part of the sucrose nonfermenting 2 (SNF2) chromatin-remodeling complex. In interphase, ATRX localizes to pericentromeric heterochromatin, contributing to DNA double-strand break repair, DNA replication, and telomere maintenance. During mitosis, most ATRX proteins are removed from chromosomal arms, leaving a pool near the centromere region in mammalian cells, which is critical for accurate chromosome congression and sister chromatid cohesion protection. However, the function and localization mechanisms of ATRX at mitotic centromeres remain largely unresolved.
The clustered regularly interspaced short palindromic repeats with CRISPR-associated protein 9 (CRISPR-Cas9) system and overexpression approaches were employed alongside immunofluorescence to investigate the mechanism of ATRX localization at the centromere. To study the binding mechanism between ATRX and heterochromatin protein 1 (HP1), both full-length and truncated mutants of hemagglutinin (HA)-ATRX were generated for co-immunoprecipitation and glutathione S-transferase (GST)-pull assays. Wild-type ATRX and HP1 binding-deficient mutants were created to investigate the role of ATRX binding to HP1 during mitosis, with the Z-Leu-Leu-Leu-al (MG132) maintenance assay, cohesion function assay, and kinetochore distance measurement.
Our research demonstrated that HP1α, HP1β, and HP1γ facilitate the positioning of ATRX within the mitotic centromere area through their interaction with the first two [P/L]-X-V-X-[M/L/V] (PxVxL)motifs at the N-terminus of ATRX. ATRX deficiency causes aberrant mitosis and decreased centromeric cohesion. Furthermore, reducing Wapl activity can bypass the need for ATRX to protect centromeric cohesion. These results provide insights into the mechanism of ATRX's centromeric localization and its critical function in preserving centromeric cohesion by reducing Wapl activity in human cells.
X连锁α地中海贫血/智力发育迟缓综合征（ATRX）是蔗糖非发酵2（SNF2）染色质重塑复合物的一部分。在间期，ATRX定位于着丝粒周围异染色质，有助于DNA双链断裂修复、DNA复制和端粒维持。在有丝分裂期间，大多数ATRX蛋白从染色体臂上移除，在哺乳动物细胞的着丝粒区域附近留下一部分，这对于准确的染色体排列和姐妹染色单体黏连保护至关重要。然而，ATRX在有丝分裂着丝粒处的功能和定位机制仍 largely未得到解决。
采用成簇规律间隔短回文重复序列与CRISPR相关蛋白9（CRISPR-Cas9）系统及过表达方法，并结合免疫荧光来研究ATRX在着丝粒处的定位机制。为了研究ATRX与异染色质蛋白1（HP1）之间的结合机制，构建了血凝素（HA）-ATRX的全长和截短突变体用于共免疫沉淀和谷胱甘肽S-转移酶（GST）-pull-down实验。创建野生型ATRX和HP1结合缺陷突变体，通过Z-亮氨酸-亮氨酸-亮氨酸-丙氨酸（MG132）维持实验、黏连功能实验和动粒距离测量来研究有丝分裂期间ATRX与HP1结合的作用。
我们的研究表明，HP1α、HP1β和HP1γ通过与ATRX N端的前两个[P/L]-X-V-X-[M/L/V]（PxVxL）基序相互作用，促进ATRX在有丝分裂着丝粒区域内的定位。ATRX缺陷会导致有丝分裂异常和着丝粒黏连减少。此外，降低Wapl活性可以绕过ATRX保护着丝粒黏连的需求。这些结果为ATRX在着丝粒处的定位机制及其通过降低人类细胞中Wapl活性来维持着丝粒黏连的关键功能提供了见解。
