ABSTRACT

The Origin Replication Complex subunit 4 (ORC4) is one in six subunits of the Origin Replication Complexes (ORCs) which is essential for initiating licensing at DNA replication origins and recruiting adaptor molecules necessary for various cellular processes. Previously, we reported that ORC4 also plays a vital role in polar body extrusion (PBE) during oogenesis in which half the chromosomes are extruded from the oocyte. We hypothesized that ORC4 might play a broader role in chromatin elimination. We tested its role in enucleation during the development of erythrocytes. Murine erythroleukemia (MEL) cells can be propagated in culture indefinitely and can be induced to enucleate their DNA by treatment with Vacuolin-1, thereby mimicking normal erythrocyte enucleation. We found that ORC4 appeared around the nuclei of the MEL cells with Vacuolin-1 treatment, gradually increasing in thickness before enucleation. We then tested whether ORC4 was required for MEL enucleation by down regulating ORC4 with siRNA-ORC4 during Vacuolin-1 treatment and found that this prevented MEL enucleation. These data are consistent with the model that ORC4 is required for erythroblast enucleation just as it is for oocyte PBE. They suggest a new model in which ORC4 expression is a marker for the initiation to the enucleation pathway.

摘要

起源复制复合体亚基 4 (ORC4) 是起源复制复合体 (ORC) 的六分之一，对于在 DNA 复制起点启动许可和招募各种细胞过程所需的适配器分子至关重要。以前，我们报道了 ORC4 在卵子发生过程中的极体挤压 (PBE) 中也起着至关重要的作用，其中一半的染色体从卵母细胞中挤压出来。我们假设 ORC4 可能在染色质消除中发挥更广泛的作用。我们测试了它在红细胞发育过程中去核中的作用。鼠红白血病 (MEL) 细胞可以在培养物中无限繁殖，并且可以通过用 Vacuolin-1 处理来诱导其 DNA 去核，从而模拟正常的红细胞去核。我们发现 ORC4 出现在用 Vacuolin-1 处理的 MEL 细胞核周围，在去核前厚度逐渐增加。然后，我们通过在 Vacuolin-1 处理期间用 siRNA-ORC4 下调 ORC4 来测试 MEL 去核是否需要 ORC4，并发现这阻止了 MEL 去核。这些数据与 ORC4 是有核细胞去核所需的模型一致，就像卵母细胞 PBE 一样。他们提出了一种新模型，其中 ORC4 表达是去核途径起始的标记。这些数据与 ORC4 是有核细胞去核所需的模型一致，就像卵母细胞 PBE 一样。他们提出了一种新模型，其中 ORC4 表达是去核途径起始的标记。这些数据与 ORC4 是有核细胞去核所需的模型一致，就像卵母细胞 PBE 一样。他们提出了一种新模型，其中 ORC4 表达是去核途径起始的标记。