The role of ORC4 in enucleation of Murine Erythroleukemia (MEL) cells is similar to that in oocyte polar body extrusion

Abstract

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.